STAT6 inhibitors

ABSTRACT

The present disclosure provides compounds that are useful for inhibiting the STAT6 pathway. Also provided are related pharmaceutical compositions and methods of using the compounds. In some embodiments, the compounds may be used to treat a disease such as, e.g., an allergic lung disease, allergic rhinitis, chronic pulmonary obstructive disease, or a cancer.

This application is a divisional of U.S. application Ser. No.14/889,802, filed Nov. 6, 2015, which is national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2014/037342, filed May 8, 2014, which claims the benefit of U.S.Provisional Patent Application No. 61/821,181, filed May 8, 2013, theentirety of each of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of medicinalchemistry and medicine. More particularly, it concerns STAT6 inhibitors.

2. Description of Related Art

Asthma patients have elevated levels of the cytokines interleukin 4(IL-4) and IL-13 in their airways, which result in mucus production,airway hyperresponsiveness (AHR), eosinophil recruitment, T-Helper cell2 (Th2) activation, resulting in immunoglobulin class switching to IgE,and inflammation (reviews 1-3). These two cytokines signal through acommon receptor, the alpha chain of the IL-4 receptor (IL-4Rα). Oncytokine binding, tyrosine residues on the receptor are phosphorylatedby JAK1, JAK3, or Tyk2. Signal transducer and activator of transcription6 (STAT6), via its Src homology 2 (SH2) domain, is recruited to thephosphotyrosine residues and is then phosphorylated on Tyr641. STAT6then dimerizes via reciprocal SH2 domain-pTyr641 interactions,translocates to the nucleus, and participates in the expression of genesleading to asthma and airway hyperresponsiveness (AHR). Elevated STAT6levels have been found in the bronchial epithelium of asthma patients(Mullings et al. 2001). Stat6 knockout mice do not develop airwayhyperresponsiveness (AHR) or lung pathology associated with asthmaregardless of the asthmatic stimuli applied (Darcan-Nicolaisen et al.2009). Taken together, these results support the hypothesis thatinhibiting the activity of STAT6 is a beneficial modality for asthmatreatment (Kasaian, M. T. et al. 2008; Popescu, F. D. 2003; Chiba, Y. etal. 2009; McCusker, C. T. et al. 2007; Nagashima, S. et al. 2009;Nagashima, S. et al. 2008; Nagashima, S. et al. 2007; Ohga, K. et al.2008; Stolzenberger, S. et al. 2001; Oh, C. K. et al. 2010).

STAT6 activity has been inhibited by small molecules (Nagashima, S. etal. 2009; Nagashima, S. et al. 2008; Nagashima, S. et al. 2007; Ohga, K.et al. 2008; Chiba, Y. et al. 2009), siRNA (Darcan-Nicolaisen, Y. et al.2009), decoy oligonucleotides,¹⁵ and antibodies (Walsh, G. M. 2012;Blease, K. 2008). Nagashima et al. identified a small molecule hit byscreening a company library for the ability to inhibit a STAT6 reportergene (Nagashima, S. et al. 2007), an assay that drove lead optimizationleading to AS 1571499 (FIG. 2). Further development led to AS 1617612,also known as YM 341619 (Nagashima, S. et al. 2008), and then to AS1810722 (Nagashima et al. 2009). Zhou et al. screened libraries andidentified (R)-76 and its synthetic derivative (R)-84 that bind to STAT6and prevent phosphorylation on Tyr641 (FIG. 2). To date, none of thesematerials have proved to be effective at treating STAT6-mediateddiseases.

A number of potential phosphopeptide compounds have been prepared butfew have the potential to advance to a clinical drug candidate.Stolzenberger et al.; Stolzenberger, S. et al. 2001, prepared aphosphopeptide derived from Tyr631 of IL-4Rα, a docking site for STAT6,coupled to the antennapedia cell penetration sequence (AP/STAT6BP) (FIG.3). IL-4 stimulation of STAT6 phosphorylation in RAMOS cells wasinhibited at 5 and 10 M, but recovered at 30 min. McCusker, et. al.(2007), reported that STAT-6-IP, a phosphopeptide derived from thephosphorylation site of STAT6, Tyr641, attached to a cell penetrationsequence from TAT PT4 protein transduction domain (STAT-6-IP), inhibitedin vitro IL-4 and IL-13 expression from splenocytes from mice challengedwith ovalbumin (OVA) (FIG. 3). Importantly, in vivo intranasaladministration inhibited OVA-induced lung inflammation and mucusproduction, eosinophil migration and AHR. The same group recentlyreported that intranasal administration of STAT-6-IP inhibited the samesymptoms in a mouse asthma model induced by ragweed pollen (Wang, Y. etal. 2011). However, these materials are not likely to become commercialproducts for the treatment of asthma.

A few potential small molecular peptide mimetics have been explored thatshowed promise as potential STAT6 inhibitors. Small molecule peptidemimetics that target the SH2 domain of STAT6 have been reported in U.S.Pat. No. 6,426,331 and PCT Patent Application WO2001/083517. Althoughextensive structure-affinity relationship studies were reported, thesynthesis of only one compound which was effective to inhibit STAT6phosphorylation in intact cells was described (PM-241H, ournomenclature, FIG. 4). However, the synthesis of the compound wascomplicated and resulted in poor yields. Furthermore, that compound wasnot tested for any biological activity relating to STAT6. Clearly, thereis a need for new STAT6 inhibitors.

SUMMARY OF THE INVENTION

The present invention overcomes limitations in the prior art byproviding in some aspects compounds that can inhibit STAT6 and/or STAT5.In some embodiments, one or more of the compounds may be included in apharmaceutical composition and used to treat a disease such as, e.g.,asthma, airway hyperresponsiveness (AHR), an allergic disease, anallergic lung disease, allergic rhinitis, or chronic rhinosinusitis. Insome embodiments, a STAT6 inhibitor of the present invention may be usedto selectively inhibit STAT6 in a mammalian subject, such as a human, totreat an allergic lung disease. In some embodiments, a compound of thepresent invention may be used to treat a cancer.

An aspect of the present invention relates to a compound of the formula:

wherein: the bond between carbons 1 and 2 is a single or double bond; R₁is phosphate, —OP(O)(OR₁₀)(OR_(10′)),-alkyl_((C≤6))-P(O)(OR₁₀)(OR_(10′)), or a substituted version of any ofthese groups; wherein R₁₀ and R_(10′) are each independently hydrogen,alkyl_((C≤6)), aryl_((C≤8)), aralkyl_((C≤12)),-alkyl_((C≤6))-O—C(O)-alkyl_((C≤6)), -alkyl_((C≤6))-O—C(O)-aryl_((C≤8)),or

wherein m=0-8; wherein X is —CH₂—, —O—, —S—, or —NH—; provided that R₁₀and R_(10′) are not both hydrogen; R₂ is hydrogen or R₂ is takentogether with R₁₁ as provided below; R₃, R₅, R₆, and R₇ are eachindependently hydrogen, unsubstituted alkyl_((C≤6)), or substitutedalkyl_((C≤6)), or (R₇ and R₈) are taken together as provided below, or(R₇, R₈, and R₉) are taken together as provided below; R₄ is hydrogen or—N(R₁₁)R₁₂; wherein: R₁₁ is hydrogen, alkyl_((C≤6)), aryl_((C≤8)),acyl_((C≤6)), or a substituted version of any of these groups, or R₁₁ istaken together with R₂ as provided, below; R₁₂ is hydrogen,alkyl_((C≤6)), acyl_((C≤6)), or R₁₂ is taken together with R₁₁ asprovided below; R₈ is hydrogen, unsubstituted alkyl_((C≤6)), substitutedalkyl_((C≤6)), unsubstituted aryl_((C≤8)), substituted aryl_((C≤8)), anamino acid, -alkanediyl_((C≤6))-C(O)NX₁X₂, —CH₂—C(O)NX₁X₂, wherein X₁and X₂ are each independently alkyl_((C≤6)), aryl_((C≤12)), or asubstituted version of either of these groups,

Nor R₈ is taken together with R₇ as provided below, or R₈ is takentogether with R₇ and R₉ as provided below, or R₈ is taken together withR₉ as provided below; R₉ is hydrogen, unsubstituted alkyl_((C≤6)),substituted alkyl_((C≤6)), unsubstituted aryl_((C≤8)), substitutedaryl_((C≤8)), an amino acid, -alkanediyl_((C≤6))-C(O)NX₁X₂,—CH₂—C(O)NX₁X₂, wherein X₁ and X₂ are each independently alkyl_((C≤6)),aryl_((C≤12)), or a substituted version of either of these groups,

or R₉ is taken together with R₇ and R₈ as provided below, or R₉ is takentogether with R₈ as provided below; provided that when R₄ is —N(R₁₁)R₁₂and (R₂ and R₁₁) are taken together, the compound is further defined byformula IA:

provided that when R₄ is —N(R₁₁)R₁₂ and (R₁₁ and R₁₂) are takentogether, the compound is further defined by formula IB:

wherein: R₁₃ and R₁₄ are each independently hydrogen or oxo; and n is 1,2, 3, 4, or 5; provided that when R₇ and R₈ are taken together, thecompound is further defined by formula IC:

provided that when R₇, R₈, and R₉ are taken together, the compound isfurther defined by formula ID or formula IE:

wherein: R₁₅ is hydrogen or —C(O)NR₁₆R₁₇; wherein: R₁₆ and R₁₇ are eachindependently hydrogen, alkyl_((C≤6)), aryl_((C≤8)), or a substitutedversion of any of these groups; R₁₈ is hydrogen,-alkenediyl_((C≤6))-aryl_((C≤8)), aralkyl_((C≤12)), —C(O)-alkyl_((C≤6)),—C(O)-heterocycloalkyl_((C≤12)), —C(O)-heteroaryl_((C≤12)),

or —C(O)NR₁₉R₂₀; wherein: R₁₉ and R₂₀ are each independently hydrogen,alkyl_((C≤6)), aryl_((C≤8)), or a substituted version of either of thesegroups; o is 1, 2, or 3; and p is 1, 2, 3, 4, or 5;provided that when R₈ and R₉ are taken together, the compound is furtherdefined by formula IF:

wherein if R₁₈ is —C(O)NR₁₉R₂₀ and R₁₉ is aryl_((C≤8)), then R₃ is nothydrogen; or a pharmaceutically acceptable salt thereof.

The compound may have the formula I. The compound may have the formulaIA. The compound may have the formula IB. The compound may have theformula IC. The compound may have the formula ID. The compound may havethe formula IE. The compound may have the formula IF. In someembodiments, R₁₈ is —C(O)NR₁₉R₂₀, R₁₉ is aryl_((C≤8)), and R₃ is —CH₃.In some embodiments, the bond between carbons 1 and 2 is a double bond.In some embodiments, R₁ is phosphate. In some embodiments, R₁ is—CF₂—P(O)(OR₁₀)(OR_(10′)). In some embodiments, R₁₀ or R_(10′) is—CH₂OC(O)C(CH₃)₃. In some embodiments, R₁₀ and R_(10′) is—CH₂OC(O)C(CH₃)₃. In some embodiments, m=1-8; wherein X is —CH₂—, —O—,—S—, or —NH—. In some embodiments, if m equals then X is —CH₃, —OH, —SH,or —NH₂. In some embodiments, R₂ is hydrogen. In some embodiments, R₃ ishydrogen. In some embodiments, R₃ is alkyl_((C≤6)). In some embodiments,R₃ is methyl. In some embodiments, R₄ is hydrogen. In some embodiments,R₄ is —N(R₁₁)R₁₂. In some embodiments, R₁₁ is hydrogen. In someembodiments, R₁₂ is hydrogen. R₁₂ may be alkyl_((C≤6)). R₁₂ may bearyl_((C≤6)). R₁₂ may be acyl_((C≤6)). R₁₃ may be hydrogen. R₁₃ may beoxo. R₁₄ may be hydrogen. R₁₄ may be oxo. In some embodiments, n is 1.In some embodiments, n is 2. In some embodiments, n is 3. In someembodiments, R₅ is hydrogen. In some embodiments, R₅ is analkyl_((C≤6)). In some embodiments, R₅ is methyl. R₆ may be hydrogen. R₆may be an alkyl_((C≤6)). R₆ may be methyl. R₇ may be an alkyl_((C≤6)).R₇ may be methyl. In some embodiments, R₅, R₆, and R₇ are hydrogen ormethyl. In some embodiments, R₅, R₆, and R₇ are hydrogen. In someembodiments, R₅, R₆, and R₇ are methyl. In some embodiments, R₈ ishydrogen. In some embodiments, R₈ is trans to the carbonyl. In someembodiments, R₉ is alkyl_((C≤6)). R₉ may be heterocycloalkyl_((C≤12)).R₉ may be an amino acid. R₉ may be

In some embodiments, R₉ is

In some embodiments, R₉ is

In some embodiments, R₈ or R₉ are -alkanediyl_((C≤6))-C(O)NX₁X₂ or—CH₂—C(O)NX₁X₂, wherein X₁ and X₂ are each independently alkyl_((C≤6)),aryl_((C≤12)), or a substituted version of either of these groups andthe alkanediyl_((C≤6)) is unsubstituted or substituted. In someembodiments, the alkanediyl_((C≤6)) is —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—.The alkanediyl_((C≤6)) may be —CH₂—. In some embodiments, X₁ isaryl_((C≤12)) or substituted aryl_((C≤12)). In some embodiments, X₁ isaryl_((C≤10)) or substituted aryl_((C≤10)). In some embodiments, X₁ isaryl_((C≤8)) or substituted aryl_((C≤8)). In some embodiments, X₁ isaryl_((C6)) or substituted aryl_((C6)). X₁ may be phenyl. In someembodiments, X₂ is alkyl_((C≤6)) or substituted alkyl_((C≤6)). X₂ may bemethyl. In some embodiments, R₉ is

R₉ may be cis to the carbonyl. R₁₅ may be —C(O)NR₁₆R₁₇. R₁₆ may be transto the carbonyl. R₁₆ may be alkyl_((C≤6)). R₁₆ may be methyl. R₁₇ may becis to the carbonyl. R₁₇ may be aryl_((C≤8)). R₁₇ may be phenyl. R₁₈ maybe alkenediyl_((C≤6))-aryl_((C≤8)). R₁₈ may be —C(H)C(H)CH₂CH₂C₆H₅ or—C(H)C(CH₃)C₆H₅. R₁₈ may be aralkyl_((C≤12)). R₁₈ may be —(CH₂)₄—C₆H₅.R₁₈ may be —C(O)-heterocycloalkyl_((C≤12)). The heterocycloalkyl may beselected from piperidine, N-methylpiperazine, and morpholine. Theheterocycloalkyl may be selected from pyrrolidine, piperidine, andazepane. R₁₈ may be

In some embodiments, R₁₈ is

In some embodiments, R₁₈ is

“o” may be 1, 2, or 3. R₁₈ may be —C(O)NR₁₉R₂₀. R₁₉ may be trans to thecarbonyl. R₂₀ may be cis to the carbonyl.

In some embodiments, the compound is further defined as:

or a pharmaceutically acceptable salt thereof. In some embodiments, thecompound is PM-43I or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention relates to a compound of theformula:

or a pharmaceutically acceptable salt thereof. In some embodiments, thecompound is not PM-287H or PM-300H-A. In some embodiments, a genericformula describing compounds of the present invention may excludePM-241H. In other embodiments, the compound is PM-287H or PM-300H-A.

Another aspect of the present invention relates to a compound of theformula:

wherein: R₂₁ is phosphate, —OP(O)(OR₁₀)(OR_(10′)),-alkyl_((C≤6))-P(O)(OR₁₀)(OR_(10′)), or a substituted version of any ofthese groups; wherein R₁₀ and R_(10′) are each independently hydrogen,alkyl_((C≤6)), aryl_((C≤8)), aralkyl_((C≤12)),alkyl_((C≤6))-O—C(O)-alkyl_((C≤6)), alkyl_((C≤6))-O—C(O)-aryl_((C≤8)),or

wherein m=0-8; wherein X is —CH₂—, —O—, —S—, or —NH—; provided that R₁₀and R_(10′) are not both hydrogen; R₂₂ is hydrogen or alkyl_((C≤6)); R₂is hydrogen, alkyl_((C≤12)), substituted alkyl_((C≤12)), aryl_((C≤12)),or substituted aryl_((C≤12)); R₂₄ is aryl_((C≤12)) or substitutedaryl_((C≤12)); provided that when R₂₂ is hydrogen then R₂₃ and R₂₄ arenot both phenyl or iodophenyl; or a pharmaceutically acceptable saltthereof. R₂₁ may be -alkyl_((C≤6))-P(O)(OR₁₀)(OR_(10′)) or substituted-alkyl_((C≤6))-P(O)(OR₁₀)(OR_(10′)). In some embodiments, R₂₁ is—CF₂—P(O)(OCH₂OC(O)C(CH₃)₃)₂. R₂₂ may be hydrogen. R₂₃ may bearyl_((C≤12)). R₂₃ may be phenyl. R₂₄ may be aryl_((C8-C12)). R₂₄ may bebiphenyl. In some embodiments, compound has the formula:

or a pharmaceutically acceptable salt thereof.

Yet another aspect of the present invention relates to a pharmaceuticalcomposition comprising a compound of the present invention and anexcipient. The pharmaceutical composition may be formulated for oral,intravenous, intranasal, or inhalational administration. In someembodiments, the pharmaceutical composition is comprised in a nebulizer,an inhaler, or a nasal spray. The pharmaceutical composition may furthercomprise a bronchodialator. In some embodiments, the bronchodialator isa long-acting β2 agonist (e.g., salmeterol, carvedilol, formoterol).

Another aspect of the present invention relates to a method of treatingan allergic or inflammatory disease in a subject comprisingadministering to the subject a therapeutically effective amount of acompound of the present invention to the subject. The disease may be alung disease such as, e.g., asthma, airway hyperresponsiveness (AHR), anallergic disease, allergic rhinitis, emphysema, chronic obstructivepulmonary disease (COPD), reactive airway disease, chronicrhinosinusitis, or essentially any other disease of the upper or lowerairways that produces airflow obstruction. The method may furthercomprise inhibition of a second STAT protein. The other STAT protein maybe STAT5 or STAT3. The method may further comprise administering to thesubject a second therapeutic compound. The second therapeutic may beadministered simultaneously or concurrently with the compound orsequentially to the compound. The second therapeutic may be in the samepharmaceutical preparation as the compound or a different pharmaceuticalfrom the compound. The second therapeutic compound may be abronchodialator (e.g., short-acting β-2 agonist, a long-acting β2agonist, or an anticholinergic), an anti-inflammatory steroid, anantihistamine, or an anti-fungal antibiotic or any combination of theseagents. The anti-inflammatory steroid may be a corticosteroid such as,e.g., fluticasone, beclomethasone, etc. In some embodiments, the secondtherapeutic compound is a non-inhaled therapeutic agent such as, e.g., aleukotriene receptor modifier (e.g., montelukast), and anti-IgE antibody(e.g., omalizumab), magnesium, theophylline, an allergen immunotherapy,or an oral or intravenous corticosteroid (e.g., prednisone,methylpredisolone). It is anticipated that, in some embodiments, asecond therapeutic may not be needed to treat the disease, and in someembodiments a STAT6 inhibitor of the present invention may beadministered to a subject as a monotherapy.

In some embodiments, the second therapeutic compound is a β2 adrenergicreceptor (β2-AR) agonist such as, e.g., salmeterol, formoterol,carvedilol, salbutamol, nadolol, albuterol, olodaterol or indacaterol.Without wishing to be bound by any theory, it is anticipated that acombination therapy comprising a STAT6 inhibitor of the presentinvention and a β2 adrenergic receptor agonist (e.g., a long-acting betaagonist such as salmeterol, carvedilol, or formoterol, or an ultralong-acting beta agonist such as indacaterol or olodaterol) may beadministered as a combination therapy, which may result in reducing ordecreasing the possibility of one or more adverse effects or toxic sideeffects of the β2 adrenergic receptor agonist (e.g., a side-effect ofthe long-acting beta agonist that promotes STAT6 activation orprogression of an allergic airway disease). In some embodiments, a STAT6inhibitor of the present invention may be administered to the subject ina single pharmaceutical preparation (e.g., a metered dose inhaler, ametered dose nose spray, etc.) with the β2 adrenergic receptor agonist.In some embodiments, the STAT6 inhibitor may be administeredsimultaneously or sequentially with the β2-AR agonist to a subject. TheSTAT6 inhibitor may be in the same pharmaceutical preparation as theβ2-AR agonist or a different pharmaceutical preparation than the β2-ARTagonist.

Yet another aspect of the present invention relates to a method ofinhibiting STAT6 in a subject comprising administering to the subject acompound of the present invention to the subject in an amount effectiveto inhibit STAT6. The subject may be a mammal such as, e.g., a human.The human may have an allergic lung disease or a cancer. The compoundmay be administered to the subject in an amount sufficient to treat theallergic lung disease or the cancer in the subject. The cancer mayexhibit increased STAT5 or STAT6 expression or activity.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed.

FIG. 1: The STAT6 pathway for asthma. This figure is adapted from Oh, etal., 2010, which is incorporated herein by reference.

FIG. 2: A number of current small molecule inhibitors of STAT6 activityare shown.

FIG. 3: Sequences of cell penetrating phosphopeptide inhibitors ofSTAT6. The sequences can be found in Stolzenberg, et al., 2011,McCusker, et al., 2007, and McClusker, et al., 2011, both of which areincorporated herein by reference.

FIG. 4: Phosphopeptide mimetic prodrug candidate which was described byU.S. Pat. No. 6,426,331 and WO 2001/083517, both of which areincorporated herein by reference. The derivative PM-242H was designedand prepared by the inventors as an analog to the previously disclosedPM-241H.

FIG. 5: Schematic description of the mechanism by which the POM groupacts as a prodrug increasing the cell permeability of the compound.

FIG. 6: Gel images of the effects of PM-242H on STAT6 phosphorylationand β-actin compared to the effects of control compound DLPC.

FIG. 7: Gel images of the effects of several inhibitors described hereinon STAT6 phosphorylation and β-actin. Each inhibitor shows the gel ofthe concentration of inhibitor and the percent inhibition observed forthe compound at a range of concentrations from 0 to 5 μM.

FIG. 8: Dose-response inhibition of STAT6 phosphorylation and effect onproliferation of Beas-2B cells.

FIGS. 9A-B: In vitro selectivity study of inhibitors 43-I, 63-I, 74-I,80-I, 81-I, and 86-I in MDA-468-MB (FIG. 9A) and the effects onproliferations (FIG. 9B).

FIGS. 10A-10D: Long-acting beta agonists enhance the expression ofallergic airway disease. FIG. 10A: Experimental protocol. FIG. 10B:Airway hyperreactivity as assessed in C57BL/6 mice or genotype matchedmice deficient in the beta 2 adrenergic receptor (β₂-AR; β₂-AR^(−/−))challenged with either control vehicle (PBS) or A. niger conidia (AN)and treated with either dilauroylphosphatidyl-choline liposomes alone(DLPC), or DLPC and the β₂-AR ligands salmeterol (Sx), formoterol (Fx)or carvedilol (Cv) as indicated. FIG. 10C: Airway hyperreactivity inmice challenged as in b, but comparing wild type mice to genotypematched mice deficient in beta arrestin 2 (βarr2^(−/−)). FIG. 10D:Bronchoalveolar lavage fluid inflammatory cells from selected mice inFIG. 10B, including total inflammatory cells, eosinophils (Eos),macrophages (mono), neutrophils (Neut) and lymphocytes (Lym). *: P<0.05,with relevant comparisons indicated.

FIGS. 11A-H: PM-242H inhibits Th cells.

FIG. 12: Treatment with PM-242H blocks the development of allergic lungdisease.

FIGS. 13A-F: Treatment with PM-242H promotes recovery from airwayhyperresponsiveness (AHR) and leads to inhibition of mucus production.

FIG. 14: Initial screening for inhibition with a variety of disclosedinhibitors using BEAS-2B cell line.

FIGS. 15A-B: Induction model for PM-86I (FIG. 15A) and PM-43I (FIG.15B).

FIGS. 16A-B: Toxicity study of PM-86I (FIG. 16A) and PM-43II (FIG. 16B).

FIGS. 17A-C: PM-43I reverses established allergic airway disease. FIG.17A, Mice are treated intranasally with 400,000 Aspergillus nigerconidia every other day. After two weeks A. niger conidia areadministered with either DLPC or DLPC/PM-43I. Airway hyperresponsivenessis measured at week 2, 3, and 4. FIG. 17B, Airway hyperrepsonsivenessinduced by acetylcholine is revered by treatment with PM-43I. FIG. 17C,Treatment with PM-43I does not significantly affect immune cellrecruitment and does in inhibit the lung immune response to A. nigerinfection.

FIGS. 18A-C: Activity of intranasally administered PM-43I and PM-86I isrestricted to the lung. FIG. 18A, Mice are treated with STAT6 inhibitorintranasally every other day. Mice are sensitized to ovalbumin byintraperitoneal administration of OVA/Alum as indicated. In a separatecohort of mice, STAT6 inhibitors are administered 4 her prior toOVA/Alum administration. FIG. 18B, Intraperitoneal treatment withinhibitors block sensitization of Th2 cells to ovalbumin. FIG. 18C,Intranasal administration of STAT6 inhibitors has no effect on thesensisization of peripheral spleenocytes to ovalbumin.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In some aspects, compounds are provided that may be used to inhibit theactivity of a STAT6 protein. In some embodiments, the STAT6 inhibitor isadministered in a pro-drug form which is converted in vivo to the activecompound through cellular processes. In some embodiments, a compoundthat inhibits STAT6 can be used to treat a variety of diseases includingseveral respiratory diseases including asthma, airwayhyperresponsiveness (AHR), an allergic disease, allergic rhinitis,emphysema, or chronic rhinosinusitis. Such compounds are describedherein.

I. CHEMICAL DEFINITIONS

When used in the context of a chemical group: “hydrogen” means —H;“hydroxy” means —OH; “oxo” means=O; “carbonyl” means —C(═O)—; “carboxy”means —C(═O)OH (also written as —COOH or —CO₂H); “halo” meansindependently —F, —Cl, —Br or —I; “amino” means —NH₂; “hydroxyamino”means —NHOH; “nitro” means —NO₂; imino means=NH; “cyano” means —CN;“isocyanate” means —N═C═O; “azido” means —N₃; in a monovalent context“phosphate” means —OP(O)(OH)₂ or a deprotonated form thereof; in adivalent context “phosphate” means —OP(O)(OH)O— or a deprotonated formthereof; “mercapto” means —SH; and “thio” means=S; “sulfonyl” means—S(O)₂—; and “sulfinyl” means —S(O)—.

In the context of chemical formulas, the symbol “—” means a single bond,“═” means a double bond, and “≡” means triple bond. The symbol “

” represents an optional bond, which if present is either single ordouble. The symbol “

” represents a single bond or a double bond. Thus, for example, thestructure

includes the structures

As will be understood by a person of skill in the art, no one such ringatom forms part of more than one double bond. The symbol “

”, when drawn perpendicularly across a bond indicates a point ofattachment of the group. It is noted that the point of attachment istypically only identified in this manner for larger groups in order toassist the reader in rapidly and unambiguously identifying a point ofattachment. The symbol “

” means a single bond where the group attached to the thick end of thewedge is “out of the page.” The symbol “

” means a single bond where the group attached to the thick end of thewedge is “into the page”. The symbol “

” means a single bond where the conformation (e.g., either R or S) orthe geometry is undefined (e.g., either E or Z).

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.When a group “R” is depicted as a “floating group” on a ring system, forexample, in the formula:

then R may replace any hydrogen atom attached to any of the ring atoms,including a depicted, implied, or expressly defined hydrogen, so long asa stable structure is formed. When a group “R” is depicted as a“floating group” on a fused ring system, as for example in the formula:

then R may replace any hydrogen attached to any of the ring atoms ofeither of the fused rings unless specified otherwise. Replaceablehydrogens include depicted hydrogens (e.g., the hydrogen attached to thenitrogen in the formula above), implied hydrogens (e.g., a hydrogen ofthe formula above that is not shown but understood to be present),expressly defined hydrogens, and optional hydrogens whose presencedepends on the identity of a ring atom (e.g., a hydrogen attached togroup X, when X equals —CH—), so long as a stable structure is formed.In the example depicted, R may reside on either the 5-membered or the6-membered ring of the fused ring system. In the formula above, thesubscript letter “y” immediately following the group “R” enclosed inparentheses, represents a numeric variable. Unless specified otherwise,this variable can be 0, 1, 2, or any integer greater than 2, onlylimited by the maximum number of replaceable hydrogen atoms of the ringor ring system.

For the groups and classes below, the following parenthetical subscriptsfurther define the group/class as follows: “(Cn)” defines the exactnumber (n) of carbon atoms in the group/class. “(C≤n)” defines themaximum number (n) of carbon atoms that can be in the group/class, withthe minimum number as small as possible for the group in question, e.g.,it is understood that the minimum number of carbon atoms in the group“alkenyl_((C≤8))” or the class “alkene_((C≤8))” is two. For example,“alkoxy_((C≤10))” designates those alkoxy groups having from 1 to 10carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3 to 10 carbon atoms). (Cn-n′) defines both theminimum (n) and maximum number (n′) of carbon atoms in the group.Similarly, “alkyl_((C2-10))” designates those alkyl groups having from 2to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3 to 10 carbon atoms)).

The term “saturated” as used herein means the compound or group somodified has no carbon-carbon double and no carbon-carbon triple bonds,except as noted below. The term does not preclude carbon-heteroatommultiple bonds, for example a carbon oxygen double bond or a carbonnitrogen double bond. Moreover, it does not preclude a carbon-carbondouble bond that may occur as part of keto-enol tautomerism orimine/enamine tautomerism.

The term “aliphatic” when used without the “substituted” modifiersignifies that the compound/group so modified is an acyclic or cyclic,but non-aromatic hydrocarbon compound or group. In aliphaticcompounds/groups, the carbon atoms can be joined together in straightchains, branched chains, or non-aromatic rings (alicyclic). Aliphaticcompounds/groups can be saturated, that is joined by single bonds(alkanes/alkyl), or unsaturated, with one or more double bonds(alkenes/alkenyl) or with one or more triple bonds (alkynes/alkynyl).Where the term “aliphatic” is used without the “substituted” modifier,then only carbon and hydrogen atoms are present. When the term is usedwith the “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.

The term “alkyl” when used without the “substituted” modifier refers toa monovalent saturated aliphatic group with a carbon atom as the pointof attachment, a linear or branched, cyclo, cyclic or acyclic structure,and no atoms other than carbon and hydrogen. Thus, as used hereincycloalkyl is a subset of alkyl. The groups —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr or propyl), —CH(CH₃)₂ (i-Pr, ^(i)Pr or isopropyl),—CH(CH₂)₂ (cyclopropyl), —CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃(sec-butyl), —CH₂CH(CH₃)₂ (isobutyl), —C(CH₃)₃ (tert-butyl, t-butyl,t-Bu or ^(t)Bu), —CH₂C(CH₃)₃ (neo-pentyl), cyclobutyl, cyclopentyl,cyclohexyl, and cyclohexylmethyl are non-limiting examples of alkylgroups. The term “alkanediyl” when used without the “substituted”modifier refers to a divalent saturated aliphatic group, with one or twosaturated carbon atom(s) as the point(s) of attachment, a linear orbranched, cyclo, cyclic or acyclic structure, no carbon-carbon double ortriple bonds, and no atoms other than carbon and hydrogen. The groups,—CH₂— (methylene), —CH₂CH₂—, —CH₂C(CH₃)₂CH₂—, —CH₂CH₂CH₂—, and

are non-limiting examples of alkanediyl groups. The term “alkylidene”when used without the “substituted” modifier refers to the divalentgroup ═CRR′ in which R and R′ are independently hydrogen, alkyl, or Rand R′ are taken together to represent an alkanediyl having at least twocarbon atoms. Non-limiting examples of alkylidene groups include: ═CH₂,═CH(CH₂CH₃), and ═C(CH₃)₂. When any of these terms is used with the“substituted” modifier one or more hydrogen atom has been independentlyreplaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH,—OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂,—OC(O)CH₃, or —S(O)₂NH₂. The following groups are non-limiting examplesof substituted alkyl groups: —CH₂OH, —CH₂Cl, —CF₃, —CH₂CN, —CH₂C(O)OH,—CH₂C(O)OCH₃, —CH₂C(O)NH₂, —CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OC(O)CH₃, —CH₂NH₂,—CH₂N(CH₃)₂, and —CH₂CH₂Cl. The term “haloalkyl” is a subset ofsubstituted alkyl, in which one or more hydrogen atoms has beensubstituted with a halo group and no other atoms aside from carbon,hydrogen and halogen are present. The group, —CH₂Cl is a non-limitingexample of a haloalkyl. An “alkane” refers to the compound H-R, whereinR is alkyl. The term “fluoroalkyl” is a subset of substituted alkyl, inwhich one or more hydrogen has been substituted with a fluoro group andno other atoms aside from carbon, hydrogen and fluorine are present. Thegroups, —CH₂F, —CF₃, and —CH₂CF₃ are non-limiting examples offluoroalkyl groups. An “alkane” refers to the compound H-R, wherein R isalkyl.

The term “alkenyl” when used without the “substituted” modifier refersto an monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, at least one nonaromatic carbon-carbon double bond, nocarbon-carbon triple bonds, and no atoms other than carbon and hydrogen.Non-limiting examples of alkenyl groups include: —CH═CH₂ (vinyl),—CH═CHCH₃, —CH═CHCH₂CH₃, —CH₂CH═CH₂ (allyl), —CH₂CH═CHCH₃, and—CH═CH—C₆H₅. The term “alkenediyl” when used without the “substituted”modifier refers to a divalent unsaturated aliphatic group, with twocarbon atoms as points of attachment, a linear or branched, cyclo,cyclic or acyclic structure, at least one nonaromatic carbon-carbondouble bond, no carbon-carbon triple bonds, and no atoms other thancarbon and hydrogen. The groups, —CH═CH—, —CH═C(CH₃)CH₂—, —CH═CHCH₂—,and

are non-limiting examples of alkenediyl groups. When these terms areused with the “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. The groups, —CH═CHF,—CH═CHCl and —CH═CHBr, are non-limiting examples of substituted alkenylgroups. An “alkene” refers to the compound H-R, wherein R is alkenyl.

The term “alkynyl” when used without the “substituted” modifier refersto an monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, at least one carbon-carbon triple bond, and no atoms otherthan carbon and hydrogen. As used herein, the term alkynyl does notpreclude the presence of one or more non-aromatic carbon-carbon doublebonds. The groups, —C≡CH, —C≡CCH₃, and —CH₂C≡CCH₃, are non-limitingexamples of alkynyl groups. When alkynyl is used with the “substituted”modifier one or more hydrogen atom has been independently replaced by—OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃,—OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or—S(O)₂NH₂. An “alkyne” refers to the compound H-R, wherein R is alkynyl.

The term “aryl” when used without the “substituted” modifier refers to amonovalent unsaturated aromatic group with an aromatic carbon atom asthe point of attachment, said carbon atom forming part of a one or moresix-membered aromatic ring structure, wherein the ring atoms are allcarbon, and wherein the group consists of no atoms other than carbon andhydrogen. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl group (carbon number limitation permitting) attached tothe first aromatic ring or any additional aromatic ring present.Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl,(dimethyl)phenyl, —C₆H₄CH₂CH₃ (ethylphenyl), naphthyl, and themonovalent group derived from biphenyl. The term “arenediyl” when usedwithout the “substituted” modifier refers to a divalent aromatic groupwith two aromatic carbon atoms as points of attachment, said carbonatoms forming part of one or more six-membered aromatic ringstructure(s) wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen. Asused herein, the term does not preclude the presence of one or morealkyl group (carbon number limitation permitting) attached to the firstaromatic ring or any additional aromatic ring present. If more than onering is present, the rings may be fused or unfused. Non-limitingexamples of arenediyl groups include:

When these terms are used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. An “arene”refers to the compound H-R, wherein R is aryl.

The term “aralkyl” when used without the “substituted” modifier refersto the monovalent group -alkanediyl-aryl, in which the terms alkanediyland aryl are each used in a manner consistent with the definitionsprovided above. Non-limiting examples of aralkyls are: phenylmethyl(benzyl, Bn) and 2-phenyl-ethyl. When the term is used with the“substituted” modifier one or more hydrogen atom from the alkanediyland/or the aryl has been independently replaced by —OH, —F, —Cl, —Br,—I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃,—NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.Non-limiting examples of substituted aralkyls are:(3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent aromatic group with an aromatic carbon atom ornitrogen atom as the point of attachment, said carbon atom or nitrogenatom forming part of one or more aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heteroaryl group consists of no atoms other than carbon, hydrogen,aromatic nitrogen, aromatic oxygen and aromatic sulfur. As used herein,the term does not preclude the presence of one or more alkyl, aryl,and/or aralkyl groups (carbon number limitation permitting) attached tothe aromatic ring or aromatic ring system. If more than one ring ispresent, the rings may be fused or unfused. Non-limiting examples ofheteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im),isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl,pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl,triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term“N-heteroaryl” refers to a heteroaryl group with a nitrogen atom as thepoint of attachment. The term “heteroarenediyl” when used without the“substituted” modifier refers to an divalent aromatic group, with twoaromatic carbon atoms, two aromatic nitrogen atoms, or one aromaticcarbon atom and one aromatic nitrogen atom as the two points ofattachment, said atoms forming part of one or more aromatic ringstructure(s) wherein at least one of the ring atoms is nitrogen, oxygenor sulfur, and wherein the divalent group consists of no atoms otherthan carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromaticsulfur. As used herein, the term does not preclude the presence of oneor more alkyl, aryl, and/or aralkyl groups (carbon number limitationpermitting) attached to the aromatic ring or aromatic ring system. Ifmore than one ring is present, the rings may be fused or unfused.Non-limiting examples of heteroarenediyl groups include:

When these terms are used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.

The term “heterocycloalkyl” when used without the “substituted” modifierrefers to a monovalent non-aromatic group with a carbon atom or nitrogenatom as the point of attachment, said carbon atom or nitrogen atomforming part of one or more non-aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heterocycloalkyl group consists of no atoms other than carbon,hydrogen, nitrogen, oxygen and sulfur. As used herein, the term does notpreclude the presence of one or more alkyl groups (carbon numberlimitation permitting) attached to the ring or ring system. As usedherein, the term does not preclude the presence of one or more doublebonds in the ring or ring system, provided that the resulting groupsremains non-aromatic. If more than one ring is present, the rings may befused or unfused. Non-limiting examples of heterocycloalkyl groupsinclude aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl,morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl,tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term“N-heterocycloalkyl” refers to a heterocycloalkyl group with a nitrogenatom as the point of attachment. When the term “heterocycloalkyl” usedwith the “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, —S(O)₂NH₂, or —C(O)OC(CH₃)₃(tert-butyloxycarbonyl, BOC).

The term “acyl” when used without the “substituted” modifier refers tothe group —C(O)R, in which R is a hydrogen, alkyl, aryl, aralkyl orheteroaryl, as those terms are defined above. The groups, —CHO, —C(O)CH₃(acetyl, Ac), —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃, —C(O)CH(CH₃)₂, —C(O)CH(CH₂)₂,—C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)CH₂C₆H₅, —C(O)(imidazolyl) arenon-limiting examples of acyl groups. A “thioacyl” is defined in ananalogous manner, except that the oxygen atom of the group —C(O)R hasbeen replaced with a sulfur atom, —C(S)R. When either of these terms areused with the “substituted” modifier one or more hydrogen atom(including a hydrogen atom directly attached the carbonyl orthiocarbonyl group, if any) has been independently replaced by —OH, —F,—Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃,—C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or—S(O)₂NH₂. The groups, —C(O)CH₂CF₃, —CO₂H (carboxyl), —CO₂CH₃(methylcarboxyl), —CO₂CH₂CH₃, —C(O)NH₂ (carbamoyl), and —CON(CH₃)₂, arenon-limiting examples of substituted acyl groups.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples of alkoxy groups include: —OCH₃ (methoxy),—OCH₂CH₃ (ethoxy), —OCH₂CH₂CH₃, —OCH(CH₃)₂ (isopropoxy), —O(CH₃)₃(tert-butoxy), —OCH(CH₂)₂, —O-cyclopentyl, and —O-cyclohexyl. The terms“alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”,“heterocycloalkoxy”, and “acyloxy”, when used without the “substituted”modifier, refers to groups, defined as —OR, in which R is alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl,respectively. The term “alkoxydiyl” refers to the divalent group—O-alkanediyl-, —O-alkanediyl-O—, or -alkanediyl-O-alkanediyl-. The term“alkylthio” and “acylthio” when used without the “substituted” modifierrefers to the group —SR, in which R is an alkyl and acyl, respectively.When any of these terms is used with the “substituted” modifier one ormore hydrogen atom has been independently replaced by —OH, —F, —Cl, —Br,—I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃,—NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. The term“alcohol” corresponds to an alkane, as defined above, wherein at leastone of the hydrogen atoms has been replaced with a hydroxy group.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylamino groups include:—NHCH₃ and —NHCH₂CH₃. The term “dialkylamino” when used without the“substituted” modifier refers to the group —NRR′, in which R and R′ canbe the same or different alkyl groups, or R and R′ can be taken togetherto represent an alkanediyl. Non-limiting examples of dialkylamino groupsinclude: —N(CH₃)₂, —N(CH₃)(CH₂CH₃), and N-pyrrolidinyl. The terms“alkoxyamino”, “alkenylamino”, “alkynylamino”, “arylamino”,“aralkylamino”, “heteroarylamino”, “heterocycloalkylamino” and “alkylsulfonylamino” when used without the “substituted” modifier, refers togroups, defined as —NHR, in which R is alkoxy, alkenyl, alkynyl, aryl,aralkyl, heteroaryl, heterocycloalkyl, and alkylsulfonyl, respectively.A non-limiting example of an arylamino group is —NHC₆H₅. The term“amido” (acylamino), when used without the “substituted” modifier,refers to the group —NHR, in which R is acyl, as that term is definedabove. A non-limiting example of an amido group is —NHC(O)CH₃. The term“alkylimino” when used without the “substituted” modifier refers to thedivalent group ═NR, in which R is an alkyl, as that term is definedabove. The term “alkylaminodiyl” refers to the divalent group—NH-alkanediyl-, —NH-alkanediyl-NH—, or -alkanediyl-NH-alkanediyl-. Whenany of these terms is used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂. The groups—NHC(O)OCH₃ and —NHC(O)NHCH₃ are non-limiting examples of substitutedamido groups.

The term “alkylphosphate” when used without the “substituted” modifierrefers to the group —OP(O)(OH)(OR), in which R is an alkyl, as that termis defined above. Non-limiting examples of alkylphosphate groupsinclude: —OP(O)(OH)(OMe) and —OP(O)(OH)(OEt). The term“dialkylphosphate” when used without the “substituted” modifier refersto the group —OP(O)(OR)(OR′), in which R and R′ can be the same ordifferent alkyl groups, or R and R′ can be taken together to representan alkanediyl. Non-limiting examples of dialkylphosphate groups include:—OP(O)(OMe)₂, —OP(O)(OEt)(OMe) and —OP(O)(OEt)₂. When any of these termsis used with the “substituted” modifier one or more hydrogen atom hasbeen independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.

The terms “alkylsulfonyl” and “alkylsulfinyl” when used without the“substituted” modifier refers to the groups —S(O)₂R and —S(O)R,respectively, in which R is an alkyl, as that term is defined above. Theterms “alkenylsulfonyl”, “alkynylsulfonyl”, “arylsulfonyl”,“aralkylsulfonyl”, “heteroarylsulfonyl”, and “heterocycloalkylsulfonyl”are defined in an analogous manner. When any of these terms is used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂.

As used herein, a “chiral auxiliary” refers to a removable chiral groupthat is capable of influencing the stereoselectivity of a reaction.Persons of skill in the art are familiar with such compounds, and manyare commercially available.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult. “Effective amount,” “Therapeutically effective amount” or“pharmaceutically effective amount” when used in the context of treatinga patient or subject with a compound means that amount of the compoundwhich, when administered to a subject or patient for treating a disease,is sufficient to effect such treatment for the disease.

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dihydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained. This quantitative measureindicates how much of a particular drug or other substance (inhibitor)is needed to inhibit a given biological, biochemical or chemical process(or component of a process, i.e. an enzyme, cell, cell receptor ormicroorganism) by half.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this invention is not critical, so long asthe salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag HelveticaChimica Acta, 2002).

The term “pharmaceutically acceptable carrier,” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a chemical agent.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

“Prodrug” means a compound that is convertible in vivo metabolicallyinto an inhibitor according to the present invention. The prodrug itselfmay or may not also have activity with respect to a given targetprotein. For example, a compound comprising a hydroxy group may beadministered as an ester that is converted by hydrolysis in vivo to thehydroxy compound. Suitable esters that may be converted in vivo intohydroxy compounds include acetates, citrates, lactates, phosphates,tartrates, malonates, oxalates, salicylates, propionates, succinates,fumarates, maleates, methylene-bis-β-hydroxynaphthoate, gentisates,isethionates, di-p-toluoyltartrates, methanesulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexylsulfamates, quinates, esters of amino acids, and the like.Similarly, a compound comprising an amine group may be administered asan amide that is converted by hydrolysis in vivo to the amine compound.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers. Chiral molecules contain achiral center, also referred to as a stereocenter or stereogenic center,which is any point, though not necessarily an atom, in a moleculebearing groups such that an interchanging of any two groups leads to astereoisomer. In organic compounds, the chiral center is typically acarbon, phosphorus or sulfur atom, though it is also possible for otheratoms to be stereocenters in organic and inorganic compounds. A moleculecan have multiple stereocenters, giving it many stereoisomers. Incompounds whose stereoisomerism is due to tetrahedral stereogeniccenters (e.g., tetrahedral carbon), the total number of hypotheticallypossible stereoisomers will not exceed 2^(n), where n is the number oftetrahedral stereocenters. Molecules with symmetry frequently have fewerthan the maximum possible number of stereoisomers. A 50:50 mixture ofenantiomers is referred to as a racemic mixture. Alternatively, amixture of enantiomers can be enantiomerically enriched so that oneenantiomer is present in an amount greater than 50%. Typically,enantiomers and/or diastereomers can be resolved or separated usingtechniques known in the art. It is contemplated that that for anystereocenter or axis of chirality for which stereochemistry has not beendefined, that stereocenter or axis of chirality can be present in its Rform, S form, or as a mixture of the R and S forms, including racemicand non-racemic mixtures. As used herein, the phrase “substantially freefrom other stereoisomers” means that the composition contains ≤15%, morepreferably ≤10%, even more preferably ≤5%, or most preferably ≤1% ofanother stereoisomer(s).

“Substituent convertible to hydrogen in vivo” means any group that isconvertible to a hydrogen atom by enzymological or chemical meansincluding, but not limited to, hydrolysis and hydrogenolysis. Examplesinclude hydrolyzable groups, such as acyl groups, groups having anoxycarbonyl group, amino acid residues, peptide residues,o-nitrophenylsulfenyl, trimethylsilyl, tetrahydropyranyl,diphenylphosphinyl, and the like. Examples of acyl groups includeformyl, acetyl, trifluoroacetyl, and the like. Examples of groups havingan oxycarbonyl group include ethoxycarbonyl, tert-butoxycarbonyl(—C(O)OC(CH₃)₃), benzyloxycarbonyl, p-methoxybenzyloxycarbonyl,vinyloxycarbonyl, β-(p-toluenesulfonyl)ethoxycarbonyl, and the like.Suitable amino acid residues include, but are not limited to, residuesof Gly (glycine), Ala (alanine), Arg (arginine), Asn (asparagine), Asp(aspartic acid), Cys (cysteine), Glu (glutamic acid), His (histidine),Ile (isoleucine), Leu (leucine), Lys (lysine), Met (methionine), Phe(phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp(tryptophan), Tyr (tyrosine), Val (valine), Nva (norvaline), Hse(homoserine), 4-Hyp (4-hydroxyproline), 5-Hyl (5-hydroxylysine), Orn(ornithine) and β-Ala. Examples of suitable amino acid residues alsoinclude amino acid residues that are protected with a protecting group.Examples of suitable protecting groups include those typically employedin peptide synthesis, including acyl groups (such as formyl and acetyl),arylmethoxycarbonyl groups (such as benzyloxycarbonyl andp-nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups (—C(O)OC(CH₃)₃),and the like. Suitable peptide residues include peptide residuescomprising two to five amino acid residues. The residues of these aminoacids or peptides can be present in stereochemical configurations of theD-form, the L-form or mixtures thereof. In addition, the amino acid orpeptide residue may have an asymmetric carbon atom. Examples of suitableamino acid residues having an asymmetric carbon atom include residues ofAla, Leu, Phe, Trp, Nva, Val, Met, Ser, Lys, Thr and Tyr. Peptideresidues having an asymmetric carbon atom include peptide residueshaving one or more constituent amino acid residues having an asymmetriccarbon atom. Examples of suitable amino acid protecting groups includethose typically employed in peptide synthesis, including acyl groups(such as formyl and acetyl), arylmethoxycarbonyl groups (such asbenzyloxycarbonyl and p-nitrobenzyloxycarbonyl), tert-butoxycarbonylgroups (—C(O)OC(CH₃)₃), and the like. Other examples of substituents“convertible to hydrogen in vivo” include reductively eliminablehydrogenolyzable groups. Examples of suitable reductively eliminablehydrogenolyzable groups include, but are not limited to, arylsulfonylgroups (such as o-toluenesulfonyl); methyl groups substituted withphenyl or benzyloxy (such as benzyl, trityl and benzyloxymethyl);arylmethoxycarbonyl groups (such as benzyloxycarbonyl ando-methoxy-benzyloxycarbonyl); and haloethoxycarbonyl groups (such asβ,β,β-trichloroethoxycarbonyl and β-iodoethoxycarbonyl).

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

The above definitions supersede any conflicting definition in any of thereference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the invention in terms such that oneof ordinary skill can appreciate the scope and practice the presentinvention.

II. COMPOUNDS

The compounds provided by the present disclosure are shown, for example,above in the summary of the invention section and in the claims below.In some embodiments, the compound may also be described by the formula:

wherein: the bond between carbons 1 and 2 is a single or double bond; R₁is phosphate, —OP(O)(OR₁₀)(OR_(10′)),-alkyl_((C≤6))-P(O)(OR₁₀)(OR_(10′)), or a substituted version of any ofthese groups; wherein R₁₀ and R_(10′) are each independently hydrogen,alkyl_((C≤6)), aryl_((C≤8)), aralkyl_((C≤12)),alkyl_((C≤6))-O—C(O)-alkyl_((C≤6)), alkyl_((C≤6))-O—C(O)-aryl_((C≤8)),or

wherein m=0-8; wherein X is —CH₂—, —O—, —S—, or —NH—; provided that R₁₀and R_(10′) are not both hydrogen; R₂ is hydrogen or R₂ is takentogether with R₁₁ as provided below; R₃, R₅, R₆, and R₇ are eachindependently hydrogen, unsubstituted alkyl_((C≤6)), or substitutedalkyl_((C≤6)), or (R₇ and R₈) are taken together as provided below, or(R₇, R₈, and R₉) are taken together as provided below; R₄ is hydrogen or—N(R₁₁)R₁₂; wherein: R₁₁ is hydrogen, alkyl_((C≤6)), aryl_((C≤8)),acyl_((C≤6)), or a substituted version of any of these groups, or R₁₁ istaken together with R₂ as provided, below; R₁₂ is hydrogen,alkyl_((C≤6)), acyl_((C≤6)), or R₁₂ is taken together with R₁₁ asprovided below; R₈ is hydrogen, unsubstituted alkyl_((C≤6)), substitutedalkyl_((C≤6)), unsubstituted aryl_((C≤8)), substituted aryl_((C≤8)), anamino acid, -alkanediyl_((C≤6))-C(O)NX₁X₂, —CH₂—C(O)NX₁X₂, wherein X₁and X₂ are each independently alkyl_((C≤6)), aryl_((C≤12)), or asubstituted version of either of these groups,

or R₈ is taken together with R₇ as provided below, or R₈ is takentogether with R₇ and R₉ as provided below, or R₈ is taken together withR₉ as provided below; R₉ is hydrogen, unsubstituted alkyl_((C≤6)),substituted alkyl_((C≤6)), unsubstituted aryl_((C≤8)), substitutedaryl_((C≤8)), an amino acid, -alkanediyl_((C≤6))-C(O)NX₁X₂,—CH₂—C(O)NX₁X₂, wherein X₁ and X₂ are each independently alkyl_((C≤6)),aryl_((C≤12)), or a substituted version of either of these groups,

or R₉ is taken together with R₇ and R₈ as provided below, or R₉ is takentogether with R₈ as provided below; provided that when R₄ is —N(R₁₁)R₁₂and (R₂ and R₁₁) are taken together, the compound is further defined byformula IA:

provided that when R₄ is —N(R₁₁)R₁₂ and (R₁₁ and R₁₂) are takentogether, the compound is further defined by formula IB:

wherein: R₁₃ and R₁₄ are each independently hydrogen or oxo; and n is 1,2, 3, 4, or 5; provided that when R₇ and R₈ are taken together, thecompound is further defined by formula IC:

provided that when R₇, R₈, and R₉ are taken together, the compound isfurther defined by formula ID or formula IE:

wherein: R₁₅ is hydrogen or —C(O)NR₁₆R₁₇; wherein: R₁₆ and R₁₇ are eachindependently hydrogen, alkyl_((C≤6)), aryl_((C≤8)), or a substitutedversion of any of these groups; R₁₈ is hydrogen,-alkenediyl_((C≤6))-aryl_((C≤8)), aralkyl_((C≤12)), —C(O)-alkyl_((C≤6)),—C(O)-heterocycloalkyl_((C≤12)), —C(O)-heteroaryl_((C≤12)),

or —C(O)NR₁₉R₂₀; wherein: R₁₉ and R₂₀ are each independently hydrogen,alkyl_((C≤6)), aryl_((C≤8)), or a substituted version of either of thesegroups; o is 1, 2, or 3; and p is 1, 2, 3, 4, or 5; provided that whenR₈ and R₉ are taken together, the compound is further defined by formulaIF:

wherein if R₁₈ is —C(O)NR₁₉R₂₀ and R₁₉ is aryl_((C≤8)), then R₃ is nothydrogen; or a pharmaceutically acceptable salt thereof. In someembodiments, the compound of formula IAa or IAb can be produced from astarting material wherein R₁ is para and R₁ is meta, respectively, andR₂ is ortho to the point of attachment. They may be made using themethods outlined in the Examples section. These methods can be furthermodified and optimized using the principles and techniques of organicchemistry as applied by a person skilled in the art. Such principles andtechniques are taught, for example, in March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure (2007), which isincorporated by reference herein. Additionally, adjustments andmodifications to the methods can be made by those skilled in the art ofpeptide bond formation including the principles and techniques taught,by example, in Novabiochem®, Guide to the Selection of Building Blocksfor Peptide Synthesis (2008). Additionally, the following otherstructural modifications are envisioned for potential compoundscontemplated by the present invention.

Furthermore, the following core could be attached to the central ring ofthe tyrosine derivative:

In some embodiments, the compound is described by the formula:

wherein: R₂₁ is phosphate, —OP(O)(OR₁₀)(OR_(10′)),-alkyl_((C≤6))-P(O)(OR₁₀)(OR_(10′)), or a substituted version of any ofthese groups; wherein R₁₀ and R_(10′) are each independently hydrogen,alkyl_((C≤6)), aryl_((C≤8)), aralkyl_((C≤12)),alkyl_((C≤6))-O—C(O)-alkyl_((C≤6)), alkyl_((C≤6))-O—C(O)-aryl_((C≤8)),or

wherein m=0-8; wherein X is —CH₂—, —O—, —S—, or —NH—; provided that R₁₀and R_(10′) are not both hydrogen; R₂₂ is hydrogen or alkyl_((C≤6)); R₂₃is hydrogen, alkyl_((C≤12)), substituted alkyl_((C≤12)), aryl_((C≤12)),or substituted aryl_((C≤12)); R₂₄ is aryl_((C≤12)) or substitutedaryl_((C≤12)); provided that when R₂₂ is hydrogen then R₂₃ and R₂₄ arenot both phenyl or iodophenyl; or a pharmaceutically acceptable saltthereof.

Compounds of the invention may contain one or moreasymmetrically-substituted carbon or nitrogen atoms, and may be isolatedin optically active or racemic form. Thus, all chiral, diastereomeric,racemic form, epimeric form, and all geometric isomeric forms of achemical formula are intended, unless the specific stereochemistry orisomeric form is specifically indicated. Compounds may occur asracemates and racemic mixtures, single enantiomers, diastereomericmixtures and individual diastereomers. In some embodiments, a singlediastereomer is obtained. The chiral centers of the compounds of thepresent invention can have the S or the R configuration.

Chemical formulas used to represent compounds of the invention willtypically only show one of possibly several different tautomers. Forexample, many types of ketone groups are known to exist in equilibriumwith corresponding enol groups. Similarly, many types of imine groupsexist in equilibrium with enamine groups. Regardless of which tautomeris depicted for a given compound, and regardless of which one is mostprevalent, all tautomers of a given chemical formula are intended.

Compounds of the invention may also have the advantage that they may bemore efficacious than, be less toxic than, be longer acting than, bemore potent than, produce fewer side effects than, be more easilyabsorbed than, and/or have a better pharmacokinetic profile (e.g.,higher oral bioavailability and/or lower clearance) than, and/or haveother useful pharmacological, physical, or chemical properties over,compounds known in the prior art, whether for use in the indicationsstated herein or otherwise.

In addition, atoms making up the compounds of the present invention areintended to include all isotopic forms of such atoms. Isotopes, as usedherein, include those atoms having the same atomic number but differentmass numbers. By way of general example and without limitation, isotopesof hydrogen include tritium and deuterium, and isotopes of carboninclude ¹³C and ¹⁴C.

Compounds of the present disclosure may also exist in prodrug form.Since prodrugs are known to enhance numerous desirable qualities ofpharmaceuticals (e.g., solubility, bioavailability, manufacturing,etc.), the compounds employed in some methods of the invention may, ifdesired, be delivered in prodrug form. Thus, the invention contemplatesprodrugs of compounds of the present invention as well as methods ofdelivering prodrugs. Prodrugs of the compounds employed in the inventionmay be prepared by modifying functional groups present in the compoundin such a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent compound. Accordingly, prodrugsinclude, for example, compounds described herein in which a hydroxy,amino, or carboxy group is bonded to any group that, when the prodrug isadministered to a subject, cleaves to form a hydroxy, amino, orcarboxylic acid, respectively.

It should be recognized that the particular anion or cation forming apart of any salt form of a compound provided herein is not critical, solong as the salt, as a whole, is pharmacologically acceptable.Additional examples of pharmaceutically acceptable salts and theirmethods of preparation and use are presented in Handbook ofPharmaceutical Salts: Properties, and Use (2002), which is incorporatedherein by reference.

Those skilled in the art of organic chemistry will appreciate that manyorganic compounds can form complexes with solvents in which they arereacted or from which they are precipitated or crystallized. Thesecomplexes are known as “solvates.” For example, a complex with water isknown as a “hydrate.” Solvates of the compounds provided herein arewithin the scope of the invention. It will also be appreciated by thoseskilled in organic chemistry that many organic compounds can exist inmore than one crystalline form. For example, crystalline form may varyfrom solvate to solvate. Thus, all crystalline forms of the compoundsprovided herein or the pharmaceutically acceptable solvates thereof arewithin the scope of the present invention.

III. PHARMACEUTICAL PREPARATIONS

Certain of the methods set forth herein pertain to methods involving theadministration of a pharmaceutically and/or therapeutically effectiveamount of a compound of the present disclosure for purposes of treatinga disease or disorder associated with STAT6 and/or STAT5. In someembodiments, the disease or disorder is asthma, airwayhyperresponsiveness (AHR), an allergic disease, an allergic lungdisease, allergic rhinitis, or chronic rhinosinusitis. In someembodiments, an inhibitor of STAT6 and/or STAT5 of the present inventionmay be administered to a subject (e.g., a mammalian subject such as ahuman) orally, intravenously, intranasally (e.g., via a nose spray), viainhalation or aerosol delivery, or topically to the skin or other mucusmembranes (e.g., intra-rectally or by enema).

Moreover, it will be generally understood that a compound of the presentdisclosure can be provided in prodrug form, also discussed above,meaning that an environment to which a compound of the presentdisclosure is exposed alters the prodrug into an active, or more active,form. For example, one or more carboxylates on the compounds can becovered into esters which are cleaved in vivo to produce the activecompound. It is contemplated that the term “precursor” covers compoundsthat are considered “prodrugs.”

1. Pharmaceutical Formulations and Routes for Administration to Subjects

Any compound discussed herein is contemplated as comprised in apharmaceutical composition. Pharmaceutical compositions of the presentdisclosure comprise an effective amount of one or more candidatesubstances (e.g., a compound of the present disclosure) or additionalagents dissolved or dispersed in a pharmaceutically acceptable carrier.The phrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of apharmaceutical composition that contains at least one candidatesubstance or additional active ingredient will be known to those ofskill in the art in light of the present disclosure, as exemplified byRemington: The Science and Practice of Pharmacy, 21^(st) Ed. LippincottWilliams and Wilkins, 2005, incorporated herein by reference. Moreover,for animal (e.g., human) administration, it will be understood thatpreparations should meet sterility, pyrogenicity, general safety andpurity standards as required by the FDA's Center of Drug Evaluation andResearch.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,pp 1289-1329, 1990). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the therapeutic orpharmaceutical compositions is contemplated.

The candidate substance may comprise different types of carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it needs to be sterile for such routes ofadministration as injection. The present disclosure can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostaticaly,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intramuscularly,subcutaneously, subconjunctival, intravesicularlly, mucosally, buccally,transdermally, intrapericardially, intraumbilically, intraocularally,orally, locally, via inhalation (e.g., aerosol inhalation), viainjection, via infusion, via continuous infusion, via localizedperfusion bathing target cells directly, via a catheter, via eye or eardrops, via a lavage, in cremes, in lipid compositions (e.g., liposomes),or by other method or any combination of the foregoing as would be knownto one of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 1990).

In some embodiments, one or more STAT6 inhibitor of the presentinvention may be administered intranasally or to the lungs of a subject.For example, the STAT6 inhibitor may be administered in a nose spraysuch as a metered dose nose spray, an inhaler, or in a nebulizer (e.g.,a mechanical nebulizer such as a soft mist inhaler, a human powerednebulizer, or an electrical nebulizer such as vibrating mesh technology(VMT) nebulizer, a jet nebulizer, an ultrasonic wave nebulizer). Aswould be appreciated by one of skill, intranasal delivery and/ordelivery to the lungs may be particularly useful for treating a diseaseaffecting the nose or lungs such as, e.g., an allergic rhinitis or anallergic lung disease, etc.

A composition comprising a compound of the present disclosure may beformulated for topical administration, for example, in a cream asmentioned, or in an ointment, salve, spray, gel, lotion, or emulsion.The composition may be formulated for transmucosal, transepithelial,transendothelial, or transdermal administration. One example oftransdermal formulation is a patch. The composition may further comprisea chemical penetration enhancer, a membrane permeability agent, amembrane transport agent, a preservative, a surfactant, or a stabilizer,as these terms are known to those of skill in the art.

In one topical embodiment, the present disclosure can utilize a patch. Atransdermal or “skin” patch is a medicated adhesive patch that is placedon the skin to deliver a time released dose of medication through theskin and into the bloodstream. A wide variety of pharmaceuticals can bedelivered by transdermal patches. The main components to a transdermalpatch are (a) a liner to protect the patch during storage (removed priorto use); (b) the active agent; (c) an adhesive that serves to adhere thecomponents of the patch together along with adhering the patch to theskin; (d) a membrane to control the release of the drug from thereservoir and multi-layer patches; and (e) a backing that protects thepatch from the outer environment.

There are four main types of transdermal patches. Single-layerDrug-in-Adhesive patches have an adhesive layer that also contains theagent. In this type of patch the adhesive layer not only serves toadhere the various layers together, along with the entire system to theskin, but is also responsible for the releasing of the drug. Theadhesive layer is surrounded by a temporary liner and a backing.Multi-layer Drug-in-Adhesive patches are similar to the single-layersystem in that both adhesive layers are also responsible for thereleasing of the drug. The multi-layer system is different however thatit adds another layer of drug-in-adhesive, usually separated by amembrane (but not in all cases). This patch also has a temporaryliner-layer and a permanent backing. Reservoir patches are unlike theSingle-layer and Multi-layer Drug-in-Adhesive systems in that thereservoir transdermal system has a separate drug layer. The drug layeris a liquid compartment containing a drug solution or suspensionseparated by the adhesive layer. This patch is also backed by thebacking layer. In this type of system the rate of release is zero order.Matrix patches have a drug layer of a semisolid matrix containing a drugsolution or suspension. The adhesive layer in this patch surrounds thedrug layer partially overlaying it.

In another form of treatment, a topical application of a compound of thepresent disclosure is targeted at a natural body cavity such as themouth, pharynx, esophagus, larynx, trachea, pleural cavity, peritonealcavity, or hollow organ cavities including the bladder, colon or othervisceral organs. A variety of methods may be employed to affect thetopical application into these visceral organs or cavity surfaces. Forexample, the pharynx may be affected by simply oral swishing andgargling with solutions comprising a compound of the present disclosure.

In particular embodiments, the composition is administered to a subjectusing a drug delivery device. Any drug delivery device is contemplatedfor use in delivering a pharmaceutically effective amount of a compoundof the present disclosure.

The actual dosage amount of a composition of the present disclosureadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

The dose can be repeated as needed as determined by those of ordinaryskill in the art. Thus, in some embodiments of the methods set forthherein, a single dose is contemplated. In other embodiments, two or moredoses are contemplated. Where more than one dose is administered to asubject, the time interval between doses can be any time interval asdetermined by those of ordinary skill in the art. For example, the timeinterval between doses may be about 1 hour to about 2 hours, about 2hours to about 6 hours, about 6 hours to about 10 hours, about 10 hoursto about 24 hours, about 1 day to about 2 days, about 1 week to about 2weeks, or longer, or any time interval derivable within any of theserecited ranges.

In certain embodiments, it may be desirable to provide a continuoussupply of a pharmaceutical composition to the patient. This could beaccomplished by catheterization, followed by continuous administrationof the therapeutic agent. The administration could be intra-operative orpost-operative.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of a compound of the present disclosure. Inother embodiments, a compound of the present disclosure may comprisebetween about 2% to about 75% of the weight of the unit, or betweenabout 25% to about 60%, for example, and any range derivable therein. Inother non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg body weight, about 50 microgram/kg body weight, about 100microgram/kg body weight, about 200 microgram/kg body weight, about 350microgram/kg body weight, about 500 microgram/kg body weight, about 1milligram/kg/body weight, about 5 milligram/kg body weight, about 10milligram/kg/body weight, about 20 milligram/kg body weight, about 50milligram/kg body weight, about 100 milligram/kg body weight, about 200milligram/kg body weight, about 350 milligram/kg body weight, about 500milligram/kg body weight, to about 1000 mg/kg body weight or more peradministration, and any range derivable therein. In non-limitingexamples of a derivable range from the numbers listed herein, a range ofabout 5 mg/kg body weight to about 100 mg/kg body weight, about 5microgram/kg/body weight to about 500 milligram/kg body weight, etc.,can be administered, based on the numbers described above. In someembodiments, a metered dose of the STAT6 inhibitor may be administeredto a subject, such as a human patient; for example about 10 μg-1 mg,about 50-500 μg, about 50-300 μg, 75-275 μg, or about 100-200 μg may beadministered to the subject (e.g., intranasally or via inhalation).

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal, or combinations thereof.

The candidate substance may be formulated into a composition in a freebase, neutral, or salt form. Pharmaceutically acceptable salts, includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, glycolic, lactic, tartaric, or mandelic acid. Saltsformed with the free carboxyl groups can also be derived from inorganicbases such as for example, sodium, potassium, ammonium, calcium orferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine, TRIS, or procaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Itmay be preferable to include isotonic agents, such as, for example,sugars, sodium chloride, or combinations thereof.

In other embodiments, one may use eye or ear drops, nasal solutions orsprays, aerosols or inhalants in the present disclosure. Suchcompositions are generally designed to be compatible with the targettissue type. In a non-limiting example, nasal solutions are usuallyaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions are prepared so that they are similarin many respects to nasal secretions, so that normal ciliary action ismaintained. Thus, in certain embodiments the aqueous nasal solutionsusually are isotonic or slightly buffered to maintain a pH of about 5.5to about 6.5. In addition, antimicrobial preservatives, similar to thoseused in ophthalmic preparations, drugs, or appropriate drug stabilizers,if required, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments the candidate substance is prepared foradministration by such routes as oral ingestion. In these embodiments,the solid composition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet. Incertain embodiments, carriers for oral administration comprise inertdiluents, assimilable edible carriers or combinations thereof. In otheraspects of the disclosure, the oral composition may be prepared as asyrup or elixir. A syrup or elixir, and may comprise, for example, atleast one active agent, a sweetening agent, a preservative, a flavoringagent, a dye, a preservative, or combinations thereof.

Dosage formulations of the present pharmaceutical compositions can beprepared by combining them with a pharmaceutically acceptable carrier,such as a slow release agent, to make either immediate or slow releaseformulations as is well known in the art. Such compositions could beused, for example, in the treatment of periodontal disease and otheroral care indications. Such pharmaceutically acceptable carriers may beeither solid or liquid in form such as, for example, cornstarch,lactose, sucrose, peanut oil, olive oil, sesame oil, propylene glycoland water. If a solid carrier is used, the dosage formulation of thepresent pharmaceutical compositions may be in, for example, powder,troche, or lozenges form. If a liquid carrier is used, the dosageformulation of the present pharmaceutical compositions may be in, forexample, soft gelatin capsule, syrup liquid suspension, emulsion, orsolution form. The dosage formulations may also contain adjuvants, suchas preserving, stabilizing, wetting or emulsifying agents, or solutionpromoters. Immediate and slow release formulations are well known in theart and have been described, for example, in U.S. Pat. No. 4,764,377(the disclosure of which is incorporated herein by reference), whichdescribes a method for treating periodontal disease by means of adelivery device placed within the periodontal pocket so that release ofa therapeutic agent occurs in the immediate vicinity of the diseaseprocess. Other means of treating periodontal disease are described inU.S. Pat. No. 5,324,756, the entire contents of which are incorporatedherein by reference.

In certain embodiments an oral composition may comprise one or morebinders, excipients, disintegration agents, lubricants, flavoringagents, or combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both.

Certain coating materials are those which dissolve at about or at leastabout a pH of 5 or above, such as at about pH 5.1, 5.2, 5.3, 5.4, 5.5,5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9,7.0 or above, such as pH of about 6.5 or above. Such coatings thereforeonly begin to dissolve when they have left the stomach and entered thesmall intestine. Accordingly, these coatings may be considered entericcoatings. A thick layer of coating is provided which will dissolve inminutes to hours, thereby allowing the capsule underneath to breakuponly when it has reached the terminal ileum or the colon. Such a coatingcan be made from a variety of polymers such as cellulose acetatetrimellitate (CAT), hydroxypropylmethyl cellulose phthalate (HPMCP),polyvinyl acetate phthalate (PVAP), cellulose acetate phthalate (CAP)and shellac. For coatings of cellulose esters, a thickness of 200-250 μmwould be suitable.

Non-limiting exemplary coating materials are methyl methacrylates orcopolymers of methacrylic acid and methyl methacrylate. Such materialsare available as EUDRAGIT™ polymers (Rohm Pharma, Darmstadt, Germany).Eudragits are copolymers of methacrylic acid and methyl methacrylate.Compositions may be based on EUDRAGIT™ L100 and Eudragit S100. EUDRAGIT™L100 dissolves at pH 6 and upwards and comprises 48.3% methacrylic acidunits per g dry substance; EUDRAGIT™ S100 dissolves at pH 7 and upwardsand comprises 29.2% methacrylic acid units per g dry substance. Certaincoating compositions are based on EUDRAGIT™ L100 and EUDRAGIT™ S100 inthe range 100 parts L100:0 parts S100 to 20 parts L100:80 parts S100. Anon-limiting exemplary range is 70 parts L100:30 parts S100 to 80 partsL100:20 parts S100. For formulations where the ratio of EUDRAGIT™L100:S100 is high, a coat thickness of the order 150-200 μm ispreferable. This is equivalent to 70-110 mg of coating for a size 0capsule. For coatings where the ratio EUDRAGIT™ L100:S100 is low, a coatthickness of the order 80-120 μm is preferable, equivalent to 30 to 60mg coating for a size 0 capsule.

It is specifically contemplated that compounds of the present disclosuremay be incorporated into the polymers that act as carriers that arenonabsorbable. Compounds of the present disclosure may be, for example,covalently bonded to such polymers. Such polymers may be, for example,the polymers mentioned above and/or the polymer tails and polymerbackbones discussed herein.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina, or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides, or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and/or the other ingredients. Inthe case of sterile powders for the preparation of sterile injectablesolutions, suspensions or emulsions, certain methods of preparation mayinclude vacuum-drying or freeze-drying techniques which yield a powderof the active ingredient plus any additional desired ingredient from apreviously sterile-filtered liquid medium thereof. The liquid mediumshould be suitably buffered if necessary and the liquid diluent firstrendered isotonic prior to injection with sufficient saline or glucose.The preparation of highly concentrated compositions for direct injectionis also contemplated, where the use of DMSO as solvent is envisioned toresult in extremely rapid penetration, delivering high concentrations ofthe active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin, or combinations thereof.

2. Combination Therapy

In order to increase the effectiveness of a compound of the presentdisclosure, a compound of the present disclosure may be combined withother efficacious drugs. It is contemplated that this type ofcombination therapy may be used in vitro or in vivo.

For example, a compound of the present disclosure may be provided in acombined amount with an effective amount of a second agent (or more) amodulation of the side effects of the other drug. This process mayinvolve administering the agents at the same time or within a period oftime wherein separate administration of the substances produces adesired therapeutic benefit. This may be achieved by contacting thecell, tissue, biofilm, or organism with a single composition orpharmacological formulation that includes two or more agents, or bycontacting the cell with two or more distinct compositions orformulations, wherein one composition includes one agent and the otherincludes another.

The compounds of the present disclosure may precede, be co-current withand/or follow the other agents by intervals ranging from minutes toweeks. In embodiments where the agents are applied separately to a cell,tissue, biofilm, or organism, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agents would still be able to exert anadvantageously combined effect on the cell, tissue or organism. Forexample, in such instances, it is contemplated that one may contact thecell, tissue or organism with two, three, four or more modalitiessubstantially simultaneously (i.e., within less than about a minute) asthe candidate substance. In other aspects, one or more agents may beadministered within or substantially simultaneously, about 1 minute,about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes,about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours,about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours,about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours,about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours,about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours,about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, about 7 days, about 8 days, about 9 days, about 10days, about 11 days, about 12 days, about 13 days, about 14 days, about15 days, about 16 days, about 17 days, about 18 days, about 19 days,about 20 days, about 21 days, about 1, about 2, about 3, about 4, about5, about 6, about 7 or about 8 weeks or more, and any range derivabletherein, prior to and/or after administering the candidate substance.

Various combination regimens of the agents may be employed. Non-limitingexamples of such combinations are shown below, wherein a compound of thepresent disclosure is “A” and a second agent is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

The second agent “B” may be a steroid, an antihistamine, abronchodilator (e.g., short-acting β-2 agonist, a long-acting β2agonist, or an anticholinergic), an anti-inflammatory steroid, or anantihistamine, or an anti-fungal antibiotic or any combination of theseagents. The anti-inflammatory steroid may be a corticosteroid such as,e.g., fluticasone or beclomethasone. In some embodiments, the secondagent is a non-inhaled therapeutic agent such as, e.g., a leukotrienereceptor modifier (e.g., montelukast), and anti-IgE antibody (e.g.,omalizumab), magnesium, theophylline, an allergen immunotherapy, or anoral or intravenous corticosteroid (e.g., prednisone,methylprednisolone). In embodiments where a compound of the presentinvention is used to treat a cancer in a subject, the second agent maybe a chemotherapy, a radiotherapy, a gene therapy, or an immunotherapy.

IV. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1: STAT6 Inhibitors

The STAT6 pathway is described in FIG. 1. FIG. 1 provides a generalbackground on the molecular pathway leading to STAT6's involvement inasthma.

A. Inhibition of STAT6 Functions with Phosphopeptides Targeting the SH2Domain

Using a modular phosphopeptide prodrug synthesis methodology describedin Mandal, et al., 2011 and Mandal, et al., 2009, both of which areincorporated herein by reference, to prepare compound PM-241H. Duringthe synthesis, the iodine of PM-241H was substituted with a phenyl groupleading to a second and more efficacious prodrug, PM-242H.

In these inhibitors, the phosphate group is replaced with thedifluoromethylphosphonate group to prevent cleavage by phosphatases andthus enhance efficacy. The negative charges are blocked withcarboxyesterase-labile pivaloyloxymethyl (POM) groups. Phosphotyrosinewas replaced with the conformationally constrained cinnamate group.Substituents on the C-terminal amide nitrogen impart affinity for theSH2 domain of STAT6. On entering the cell, the POM groups are cleavedreleasing the bis-negatively charged inhibitor for interaction with theSH2 domain. (FIG. 5).

Compound PM-241H inhibited the phosphorylation of Tyr641 of STAT6 inBeas-2B cells stimulated with IL-4 and IL-13. Partial inhibition wasobserved at 1 μM and complete inhibition occurred at 10 μM. PH-242H alsoshowed similar activity which can be seen in FIG. 6.

B. Phosphates

Select phosphate-containing STAT6 inhibitors provided herein. All IC₅₀values are shown in μM.

Compound IC₅₀ (μM) PM-9I 0.771 ± 0.29  PM-10I 2.09 ± 0.93 PM-15I  2.2 ±0.17 PM-28I  0.63 ± 0.013 PM-34I 0.28 ± 0.06 PM-60I 2.33 ± 0.5  PM-67I-A0.37 ± 0.09 PM-67I-B 0.103 ± 0.05  PM-67I-C  1.9 ± 0.84 PM-59I 0.26 ±0.09 PM-87I 0.24 ± 0.13 PM-71-A 0.05 ± 0.04 PM-71-B 0.23 ± 0.12

The following structures as phosphate esters were synthesized andassayed them for the ability to compete withFAM-Ala-pTyr-Lys-Pro-Phe-Gln-Asp-Leu-Ile-NH₂, derived from Tyr631 ofIL-4Rα, for binding to STAT6 using fluorescence polarization(FAM=5-carboxyfluorescein). The fluorescence polarization methodologywas adapted from Wu et al., 1997, which is incorporated herein byreference. Amide bond replacements, cyclic amides, and cyclic lactams inthe central part of the compound were explored.

Other Phosphate-containing STAT6 inhibitors synthesized. All IC₅₀ valuesare shown in μM.

Compound IC₅₀ (μM) PM-287H 0.073 ± 0.01 PM-286H 0.053 ± 0.01 PM-300H-A0.120 ± 0.04 PM-300H-B 0.089 ± 0.01 PM-301H 0.117 ± 0.03 PM-302H-A 0.268± 0.07 PM-70I  0.23 ± 0.14 PM-26I  0.12 ± 0.016 PM-302H-B 0.377 ± 0.09

TABLE 1 Characterization of phosphate-bearing inhibitors of STAT6 SAR ofC-terminus. MS Calcd MS Found HPLC RT Compound (M + H) (M + H) (min)PM-9I 555.2624 555.2867 24.52 PM-10I 557.2780 557.2827 25.52 PM-15I527.2311 527.2312 20.47 PM-28I 516.1900 516.1879 14.40 PM-34I 576.1900576.1874 17.47 PM-60I 558.2369 558.2347 17.20 PM-67I-A 536.2526 536.257313.53 PM-67I-B 538.2318 538.2334 10.92 PM-67I-C 551.2635 551.2683 9.47PM-59I 570.2369 570.2342 19.26 PM-87I 584.2526 584.2516 19.18 PM-71I-A598.2682 598.2690 19.42 PM-71I-B 584.2526 584.2533 17.75

C. Phosphatase-Stable, Cell-Permeable Prodrugs

In the phosphatase-stable prodrugs, cyclic amide, bicyclic amide orcaprolactams groups were incorporated into phosphatase-stable,cell-permeable prodrugs and assayed for the ability to inhibit STAT6phosphorylation in immortalized human airway cells. Synthesis wascarried out using the modular, convergent technology developed for Stat3inhibitors (Scheme I) described below and included Mandal, et al., 2011and Mandal, et al., 2009, both of which are incorporated herein byreference. In short, cinnamic acid derivatized at (a), the 4 position ofthe aromatic ring with bis-pivaloyloxymethyl phosphonodifluormethylgroups, (b), at the β or 3 position of the alkene with either a hydrogenor a methyl group, and (c), the carboxy group esterified as apentachlorophenyl or 4-nitrophenyl ester, was coupled to amines ofvarying structure.

Cell-Permeable STAT6 Inhibitors are Shown Above.

Compounds were screened at 5 μM for their ability to inhibit IL-4stimulated phosphorylation of STAT6 in Beas-2B immortalized human airwaycells. Cells were treated with prodrug for 2 hr and then stimulated withIL-4. After 30 min cells were lysed and pSTAT6 and total STAT6 weremeasured with western blots (FIG. 7). Without being bound by theory, theinhibition observed is consistent with compounds entering cells, beingstripped of the POM groups, binding to the SH2 domain of STAT6, blockingbinding to IL-4Rα and preventing phosphorylation of Tyr641. PM-73I,which has no phosphonate and thus cannot bind to the SH2 domain ofSTAT6, did not inhibit STAT6 phosphorylation indicating that thephosphonate group is required to inhibit STAT6 phosphorylation.Compounds resulting in <10% residual phosphorylation were taken on toMTS cytotoxicity assays and dose response assays. (FIG. 8). The cyclicaromatic amides PM-63I, PM-80I, PM-81I, and PM-86I were very potent withIC₅₀ values from 50-500 nM. Interestingly, the non-aromatic piperidineamide, PM-74I was also very potent with and IC₅₀ of 50 nM. Lactam PM-43Iwas not as potent, IC₅₀ was between 1 and 2.5 μM. With the exception ofPM-43I, concentrations that inhibit STAT6 phosphorylation are 20-100fold lower than those resulting in toxicity. STAT6 inhibitors wereassayed for their ability to inhibit the phosphorylation of STAT 1, STAT3, STAT 5, Akt, and FAK, all processes that depend on SH2domain-phosphotyrosine interactions (Mandal, P. K. et al. 2011). Serumstarved MDA-MB-468 breast cancer cells were treated with STAT6inhibitors for 2 hr and then were stimulated with EGF (See FIGS. 9A and9B). Most compounds did not inhibit the other processes at 5 μM.However, PM-43I and PM-63I showed significant cross reactivity withStat5.

TABLE 2 Structures and characterization, of prodrug inhibitors of STAT6.MS Calcd MS Found HPLC RT Compound (M + H) (M + H) (min) PM-12I 803.3848803.3840 35.55 PM-16I 775.3535 775.3570 34.47 PM-43I 778.3280 778.326830.56 PM-42I 838.3280 838.3302 33.41 PM-64I 820.3750 820.3764 33.92PM-74I 798.3906 798.4006 33.00 PM-63I 832.3750 832.3790 35.20 PM-86I846.3906 846.3918 34.3 PM-80I 860.4063 860.4112 35.78 PM-81I 846.3906846.3908 34.54

D. Inhibitors Decrease Development of Th Cells

The ability of PM-242H to inhibit the development of major T helpereffector subsets (Th1, Th2, Th17 cells) at the time of allergen exposurewas tested. For these experiments, mice were immunized with chicken eggovalbumin precipitated in alum over two weeks and then challengedintranasally with the spores of the fungus Aspergillus niger (AN)together with alum-free ovalbumin. The inventors prepared single cellsuspensions of splenocytes from these same mice and stimulated the Tcells in these preparations with ovalbumin to determine the precisenumber of antigen-specific Th1, Th2 and Th17 cells that developed. Thisin vitro restimulation assay showed that Th2 (IL-4-producing) and Th17(IL-17A-producing) T cells failed to develop, whereas Th1 (gammainterferon (IFN-γ))-secreting cells were unaffected by PM-242H (FIG.11F-H). In summary, treatment of mice with PM-242H suppressed thedevelopment of Th2 and Th17, but not Th1 cells. These findings areentirely consistent with the known properties of STAT6, but in additionindicate that STAT6 may be involved with the production of Th17 cells.

E. In Vivo Inhibition of Asthma

The following is included to demonstrate that this class of compoundsinhibits asthma symptoms in murine models. The synthesized PM-242Hcompound was advanced to in vivo studies. PM-242H was formulated assimple dilauroylphosphatidyl-choline liposomes and applied by intranasaladministration. This formulation demonstrated ability to 1) inhibit TH2cell development in vivo, 2) inhibit the generation of airwayhyperresponsiveness and other features of asthma-like disease in a mousemodel when given prophylactically, and 3) when given therapeutically,i.e., given after the disease phenotype has already become established.PM-242H has been shown to inhibit STAT6 phosphorylation both in vitroand in vivo. These findings with PM-242H in vivo and in vitro areentirely consistent with and explained by its ability to inhibitselectively STAT6.

F. Inhibitors Block Development of Allergic Lung Disease (InductionModel)

The same model as described in part E was used to determine the effectof PH-242H on the induction of allergic lung disease. Mice that wereimmunized with ovalbumin and challenged intranasally with ovalbumin andA. niger spores developed AHR as determined by a shift to the left ofthe acetylcholine (Ach) dose response curve as compared to PBSchallenged animals. Enzyme linked immunocell spot (ELISpot) assays werethen performed on whole lung homogenates, assaying for IL-4, gammainterferon (IFN-γ) and IL-17A-secreting cells. Relative to shamimmunized animals, both IL-17A-secreting cells and the ratio betweenIL-4 and IFN-γ-secreting cells were markedly enhanced in lungs of micethat were immunized with ovalbumin and received spores and ovalbuminintranasally. In contrast, these parameters were significantlysuppressed by PH-242H, but not DLPC alone (FIG. 11C, FIG. 11D).

G. Inhibitors Block Development of Allergic Lung Disease (ReversalModel)

In separate experiments wild type C57BL/6 mice were challenged with thespores of A. niger for two weeks after which the inventors began to givePM-242H intranasally at 50 micrograms per dose at the time of continuedfungal challenge for an additional two weeks (FIG. 13A). Addition ofPM-242H during established disease resulted in the abrogation of airwayhyperresponsiveness that otherwise persisted in sham-treated mice (FIG.13B), and reduction in goblet cell metaplasia as assessed by periodicacid-Schiff staining of lung sections (FIG. 13G). PM-242H given duringestablished disease did not, however, attenuate lung and airwayinflammation (FIG. 13C-F), and in fact the number of airway eosinophilsand lung IL-17A-secreting cells increased after PM-242H challenge (FIG.13C, FIG. 13E). Thus, when given in a therapeutic context, PM-242Hreverses the STAT6-dependent allergic airway disease features of airwayhyperresponsiveness and goblet cell metaplasia, but does not diminishestablished allergic inflammation.

As shown in FIGS. 17A-C, PM-43I reverses established allergic airwaydisease. As shown in FIGS. 18A-C, activity of intranasally administeredPM-43I and PM-86I is restricted to the lung.

H. Toxicity Studies

The issue of toxicity was addressed with the newer synthetic STAT6peptidomimetic agents 86i and 43i in two ways. First, the inventorsdetermined the effect of each inhibitor on asthma-related parameters infungus-challenged mice. At an intermediate dose of 5 micrograms, PM 86iwas maximally effective at inhibiting airway inflammation and lung IL-4responses, but intermediately effective in inhibiting airwayhyperresponsiveness (FIG. 15A). However, a 10-fold higher dose of 50micrograms, although maximally effective in inhibiting airwayhyperresponsiveness, resulted in a rebound of lung inflammation and IL-4responses to sham-treated levels (FIG. 15A). Similar trends were seenwith 43i (FIG. 15B), although the maximally effective dose was 10-foldlower than for 83i (0.5 micrograms). Second, the weights of mice weremeasured during challenge with the different doses of both STAT6inhibitors. These data indicated no toxicity with 86i, i.e., there wereno significant differences in terms of weight gain over the period ofdrug administration, but 43i did result in a reduction in weight gain atthe highest dose of 50 micrograms.

I. Methods and Materials

Mice

For all mouse experiments, female mice between the ages of 4 and 8 weekswere used. All studies were conduced in compliance with all Federal andInstitutional Animal Care and Use Committee regulations. β-arrestin 2knockout (βarr2^(−/−); 8 generations backcrossed to the C57BL/6background) and β₂ Adrenergic Receptor knockout (β₂AR^(−/−); eightgenerations backcrossed to the FVB background) mice were generated aspreviously described (Walker, J. K. et al. 2003; Nguyen, L. P. et al.2009). Balb/c, C57BL/6, and FVB wild type mice were purchased fromJackson Laboratories (Bar Harbor, Me.).

Drugs and Synthetic Reagents

Salmeterol (SX; S5068), albuterol (PHR1053), carvedilol (C3993), andnadolol (N1892) were purchased from Sigma-Aldrich (St. Louis, Mo.).1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC; 770335) was purchasedfrom Avanti Polar Lipids (Alabaster, Ala.) and used as a vehicle at a1:5 (drug:DLPC) ratio. In brief, drug and DLPC were solubilized int-butanol, frozen, lyophilized, and then placed in fine suspension bythe addition of sterile, endotoxin-free phosphate buffered saline (PBS)and sonicated at 60 Hz for 30 seconds prior to use in vitro studies andadministration to mice.

Infectious Allergic Lung Disease Model

Mice were challenged intranasally with a clinical isolate of 4×10⁵ A.niger conidia every two days for a total of 8 challenges and treatedwith 50 μg of the indicated β-agonist, liposome vehicle (DLPC) and/orPM-242H (5 or 50 μg) intranasally as indicated and the allergic airwaydisease phenotype was determined according to previously describedmethods (Porter, P. et al. 2009).

Ovalbumin-Alum Allergic Lung Disease Model

Mice were sensitized to ovalbumin precipitated in alum as described inFIG. 3 (Corry, D. B. et al. 1996). In brief, mice were vaccinatedintraperitoneally weekly with ovalbumin/alum together with PM-242H (50or 250 μg) or liposome vehicle (DLPC) for 2 consecutive weeks andallowed to rest for 1 week. Mice were then intranasally challenged withovalbumin (1 mg/mL in PBS; A5503, Sigma-Aldrich) and 4×10⁵ A. nigerconidia for 5 consecutive days after which the allergic airway diseasephenotype was assessed. Randomization was not used to assign mice tospecific treatment groups. Investigators were blind as to mouse genotypeand treatment during data collection.

Allergic Airway Disease Analysis

Allergic airway disease was assessed as previously described (Porter, P.et al. 2009). Changes in respiratory system resistance (R_(RS)) inresponse to intravenous acetylcholine challenge, bronchoalveolar lavage(BAL) fluid differential counts, and analysis of lung IL-4, IL-17A andIFN-γ producing cells by enzyme linked immunocell spot assay (ELISpot)were performed as previously described (Lee, S. H. et al. 2003;Polikepahad, S. et al. 2010).

Ovalbumin Restimulation

Splenocytes from the indicated challenge groups of naïve and sensitizedmice were assessed for antigen-specific recall cytokine responses byELISpot. In brief, spleens were removed post mortem, de-aggregated bypressing through 40 μm nylon mesh and the red blood cells were removedfrom resulting cell suspension by hypotonic lysis. The splenocytes werethen washed twice and cultured in flat-bottom wells of 96-wellmicrotiter plates that were pre-coated with capture antibodies to IL-4,interferon gamma (IFN-γ) and IL-17A. Splenocytes were added in duplicatecultures of 0.5×10⁶ cells/well that were then diluted serially two-foldto 0.015×10⁶ cells/well. Cells were cultured in the presence of media orwhole ovalbumin (1 mg/mL) overnight and plates were developed aspreviously described (Kheradmand, F. et al. 2002).

Histology

Lungs were perfused of blood by cannulating the pulmonary artery andinjecting ice cold PBS until lavage returning via the left atrium wasclear. Lungs were then inflated via the trachea with 10% formalin at 25cm water pressure and the tracheas were tied off prior to removal of thecardiopulmonary unit en bloc submerging in 10% formalin overnight. Fixedlungs were then divided into individual lobes that were then halved andembedded in paraffin. Lung sections were cut at 5 microns and stainedwith the periodic acid-Schiff (PAS) kit (395B; Sigma-Aldrich, St. Louis,Mo.).

Cell Culture

Mycoplasma-free A549 (CCL-185) cells were acquired from American TypeCulture Collection (Manassas, Va.). Cells were cultured in 50% DMEM, 50%F-12 complete media until confluent and switched to culture mediacontaining 2% FBS for at least 24 h before stimulation. Long-termcultures were initiated on confluent cells by the addition of vehicle(DLPC) or select β-agonists and blockers (salbutamol, salmeterol andnadolol) at a working concentration of 10 μM. Cells were stimulated with2 ng/mL IL-13 (213-IL/CF; R&D Systems, Minneapolis, Minn.) foridentified times and harvested for protein and mRNA. Recombinant IL-6(10 ng/mL) and IL-2 (5 ng/mL) were also added as indicated (both fromR&D Systems).

Mycoplasma-free primary human epithelial cells of the sinonasal cavitywere isolated from chronic rhinosinusitis patients and plated incomplete BEGEM on type I collagen coated plates as previously described(Shaw, J. L. et al. 2013). Confluent cultures were in the presence ofvehicle (DLPC), 10 μM salbutamol or 10 μM salmeterol for 4-5 days withmedia refreshed daily. On day 5, cells were stimulated for 24 hours with2 ng/mL recombinant human IL-13.

Western Blot

Cells or lungs were isolated and lysed in RIPA buffer (9806S; CellSignaling, Danvers, Mass.), protein was quantified using BCA ProteinAssay Reagent (23227; Thermo Fisher Scientific, Rockford, Ill.) anddenatured in Laemmli sample buffer (161-0737; Bio-Rad, Hercules, Calif.)according to manufacturer's protocol. Proteins were separated onSDS-Page gels were hand-poured using a standard electrophoresis unit(Bio-Rad, Hecules, Calif.) and transferred to a membrane using iBlot GelTransfer Device (IB 1001; Invitrogen, Grand Island, N.Y.). Membraneswere blocked in 2% FBS PBST and probed for c-Src, p-c-Src, pErk1/2,Erk1/2, p STAT6, STAT6, pSTAT3, STAT3, pSTAT5, STAT5, Shp-1, andβ-actin. Signal was detected with a ChemiDoc XRS+ system (BioRad;Hercules, Calif.).

qPCR

RNA was purified from cell culture or select lobes of lung identifiedmice and RNA using the RNeasy Mini Kit (74104; Qiagen, Valencia, Calif.)or Trizol (15596-026; Invitrogen, Grand Island, N.Y.) and cDNA made withthe TaqMan Reverse Transcription kit (N8080234; Applied Biosystems,Foster City, Calif.). Probes were acquired from Applied Biosystems(Foster City, Calif.) for 18 s, CC26 and Muc5AC and used to quantifyrelative expression with TaqMan Fast Universal PCR Master Mix (435189,Applied Biosystems, Foster City, Calif.) on a the 7500 Real-Time PCRSystem (Applied Biosystems, Foster City, Calif.).

Fungal Burden

Fungal burden of fungal challenged mice was assessed by serial dilutionof lung homogenates ( 1/10 volume) on Sabouraud agar plates (84088;Sigma Aldrich, St. Louis, Mo.) in the presence of 100 mg/mLchloramphenicol (C0378; Sigma Aldrich, St. Louis, Mo.). Plates wereincubated overnight at 37° C., assessed for total number of colonies andfungal colony forming units/lung was determined by dilution.

Statistical Analysis

Data are presented as means±standard error of means (SEM). For paired,normally distributed (log-transformed respiratory system resistance(R_(RS))) data, Student's T test was used to determine significance(P<0.05). Otherwise, group comparisons of R_(RS) data were made usingANOVA with Bonferroni's correction. All other data were compared usingeither the Mann-Whitney (2 groups) or Kruskal-Wallis (>2 groups) tests.Sample sizes for animal experiments were determined based on priorstudies in which n=4 or 5 was found to be sufficient to achievesignificance with regard to R_(RS) values in moderately polarizedtreatment groups. Data variation was similar between comparedexperimental groups.

J. Synthesis Synthesis of PM-28I

The synthesis of PM-28I was performed as follows:

Reagents and conditions: a) N-methylaniline, HBTU, DIPEA, DCM,overnight; b) TFA/DCM, 1 h; c) pentachlorophenyl(E)-3-(4-phosphorylphenyl) but-2-enoate (from Mandal et al. 2011), NMM,DMAP, NMP.

A solution of commercially availableBoc-(3S)-3-amino-1-carboxymethylcaprolactam (1.1) (0.5 g, 1.75 mmol),N-methylaniline (0.2 g, 1.75 mmol), HBTU (0.8 g, 2.1 mmol) and DIPEA(0.7 mL, 3.5 mmol) in 25 mL of dry DCM was stirred for overnight. Themixture was diluted with additional 20 mL of DCM and washed with 5% HCl(2×20 mL) solution followed by 10% NaHCO₃ (1×20 mL) and brine (1×20 mL).After drying over MgSO₄ and concentrated to dryness 1.2 was then treatedwith 95% TFA-DCM for 1 h. The solvents were removed under vacuum tilldryness and the residue was diluted with water and neutralized withaqueous NH₄OH and lyophilized to give 1.3, which was used withoutpurification. Intermediate 1.3 (50.0 mg, 0.18 mmol) was dissolved in 2.0mL of NMP and 0.1 mL of NMM and 2.0 mg of DMAP were added. To this wasadded pentachlorophenyl (E)-3-(4-phosphorylphenyl) but-2-enoate (fromMandal et al. 2011) (0.18 mmol, 90.0 mg). After 2.0 hr the mixture wasapplied to a reverse phase HPLC column and was chromatographed with agradient of acetonitrile in H₂O (0.1% TFA in both solvents) to give 42.0mg (46%) of PM-28I. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C₂₅H₃₁N₃O₇P516.1900; Found 516.1879. ¹H-NMR (600 MHz, DMSO-d₆) δ 1.47 (m, 1H),1.62-1.73 (m, 3H), 1.77 (m, 1H), 1.86 (m, 1H), 2.46 (s, 3H), 2.51 (s,2H), 3.18 (s, 3H), 3.27 (d, J=15.0 Hz, 1H), 3.62 (m, 1H), 3.9 (m, 2H),4.64 (m, 1H), 6.42 (s, 1H), 7.18 (d, J=8.6 Hz, 2H), 7.4 (m, 3H), 7.48(m, 2H), 7.53 (d, J=8.6 Hz, 2H), 8.00 (d, J=7.2 Hz, 1H). ¹³C-NMR (150MHz, DMSO-d₆): δ 16.4, 26.4, 27.4, 31.2, 36.9, 49.7, 51.1, 51.3, 119.9,120.0, 127.2, 129.7, 137.7, 147.2, 151.6, 151.7, 164.9, 167.6, 172.5.

Synthesis of PM-43I

The synthesis of PM-43I was performed as follows:

Reagents and conditions: a) Pentachlorophenyl(2E)-3-[4-[[bis[(2,2-dimethyl-1-oxopropoxy)methoxy]phosphinyl]difluoromethyl]-phenyl]but-2-enoate,(from Mandal et al. 2011), NMM, DMAP, NMP.

Intermediate 1.3 (50.0 mg, 0.18 mmol) was dissolved in 2.0 mL of NMP and0.1 mL of NMM and 2.0 mg of DMAP were added. To this was addedpentachlorophenyl(2E)-3-[4-[[bis[(2,2-dimethyl-1-oxopropoxy)methoxy]phosphinyl]difluoromethyl]-phenyl]but-2-enoate(140 mg, 0.18 mmol) (from Mandal et al. 2011). After 2.0 hr the mixturewas applied to a reverse phase HPLC column and was chromatographed witha gradient of acetonitrile in H₂O to give 38 mg (27%) of PM-43I. HRMS(ESI-TOF) m/z: [M+H]+ Calcd for C₃₈H₅₁F₂N₃O₁₀P 778.3280; Found 778.3268.¹H-NMR (600 MHz, CDCl₃) δ 1.22 (s, 18), 1.56 (m, 1H), 1.61-1.8 (m, 3H),1.9 (m, 1H), 2.0 (m, 1H), 2.12 (d, J=13.2 Hz, 1H), 2.53 (s, 3H), 3.18(m, 1H), 3.28 (s, 3H), 3.72 (m, 1H), 3.93 (m, 2H), 4.72 (m, 1H), 5.65(dd, J=5.0 Hz, 2H), 5.74 (dd, J=5.0 Hz, 2H), 6.1 (s, 1H), 7.05 (d, J=6.0Hz, 1H), 7.3 (d, J=7.7 Hz, 2H), 7.4 (m, 1H), 7.45 (m, 2H), 7.5 (d, J=8.Hz, 2H), 7.6 (d, J=8.0 Hz, 2H). ¹³C-NMR (150 MHz, CDCl₃) δ 17.5, 26.8,26.9, 27.9, 31.8, 37.6, 38.7, 51.2, 52.1, 52.4, 82.4, 82.5, 121.4,126.4, 126.5, 127.3, 128.4, 130.1, 142.7, 145.6, 149.4, 165.3, 167.7,173.5, 176.5.

Synthesis of PM-242H

Synthesis of the STAT6 antagonist PM-242H was based on previouslypublished methods (Mandal, et al., 2009, U.S. Pat. No. 6,426,331) withthe strategy summarized in the scheme below.

Reagents and conditions: i) 4-aminobiphenyl, EDC, CH₂Cl₂, rt, 12 h, 91%;ii) Ph₃Bi, Cu(OAc)₂, TEA, CH₂Cl₂, rt, 48 h, 77%; iii) a) TFA, b)Fmoc-Tle-OH, HBTU, DIPEA, rt, 12 h, 82%; iv) 20% piperidine/DMF 30 min.61%; v) 6, NMP, NMM, DMAP(cat.), 2 h, 71%.

Synthesis of Boc-prolyl-4-amidobiphenyl, 2

A solution of Boc-Proline (1, 2.0 g, 9.3 mmol), 4-aminobiphenyl (1.6 g,9.3 mmol) and EDC (2.1 g, 11.2 mmol) in 60 mL of dry CH₂Cl₂ was stirredovernight. It was then transferred to a separatory funnel with anadditional 20 mL of CH₂Cl₂ and washed with 5% HCl (2×30 mL) followed by10% NaHCO₃ (2×30 mL) and brine (1×20 mL). The organic layer was dried(MgSO₄) and concentrated under reduced pressure. Purification by silicagel column chromatography eluting with 15% EtOAc-hexane afforded thetitle products as a white solid (3.1 g, 91% yield). Calcd (M+H):367.2022; Found (M+H): 367.2351. ¹H NMR (CDCl₃, 600 MHz) δ:9.6 (s, 1H),7.51-7.63 (m, 6H), 7.44 (m, 2H), 7.34 (m, 1H), 4.54 (m, 1H), 3.32-3.68(m, 2H), 1.84-2.07 (m, 4H), 1.54 (s, 9H). ¹³C NMR (CDCl₃, 150 MHz) δ:128.9, 127.5, 127.0, 126.8, 119.9, 80.9, 47.3, 28.4.

Synthesis of N-phenyl Boc-prolyl-4-amidobiphenyl, 3

To a stirred solution of 2 (2.0 g, 5.4 mmol) in dry CH₂Cl₂ (50 mL) wasadded triphenylbismuth (3.6 g, 8.2 mmol), Cu(OAc)₂ (1.6 g, 8.2 mmol) anddry triethylamine (1.2 mL, 8.2 mmol). The reaction was monitored byHPLC. After completion of the reaction, the solvent was evaporated invacuo and the residue was diluted with ether (150 mL) and filteredthrough celite. The organic layer was washed with 5% HCl (2×30 mL)followed by brine and was dried over MgSO₄. Concentration under reducedpressure followed by purification by silica gel chromatography using 10%EtOAc-hexane afforded 3 as a white solid (1.9 g, 77% yield). Calcd(M+H): 443.2335; Found (M+H): 443.2349 ¹H NMR (CDCl₃, 600 MHz) δ:7.2-7.63 (m, 14H), 4.38 (m, 1H), 4.25 (m, 1H isomer), 3.48-3.6 (m, 2H),3.4 (m, 1H), 3.32 (m, 1H isomer), 1.8-2.1 (m, 2H), 1.65-1.75 (m, 2H),1.46 (s, 9H), 1.4 (s, 9H isomer). ¹³C NMR (CDCl₃, 150 MHz) δ: 173.2,172.8, 154.4, 153.8, 129.9, 129.3, 128.8, 128.4, 127.5, 127.1, 126.4,125.9, 79.9, 79.3, 58.00, 57.8, 47.2, 31.9, 30.4, 28.8, 28.6, 28.4,24.3, 23.4.

Synthesis of Fmoc-tert-butylglycyl-N-phenyl-prolyl-4-amidobiphenyl, 4

A solution of 3 (1.00 g, 2.25 mmol) in 5 mL of neat trifluoroacetic acid(TFA) was stirred for 1 h. Excess TFA was removed under vacuum. Theresidue was then treated with Fmoc-Tle-OH (0.8 g, 2.25 mmol), HBTU (0.85g, 2.25 mmol), DIPEA (1.2 mL, 6.7 mmol) in 50 mL of dry CH₂Cl₂overnight. The organic layer was diluted with an additional 50 mL ofCH₂Cl₂ and washed with 5% HCl (3×30 mL) followed by 10% NaHCO₃ (1×30 mL)and brine. After drying (MgSO₄) and concentration under vacuum, thecrude product was purified by silica gel chromatography using 35%EtOAc-hexane to give the desired material as white foam. Yield: 1.25 g82%. Calcd (M+H): 678.3332; Found (M+H): 678.3438. ¹H NMR (CDCl₃, 600MHz) δ: 7.78 (m, 2H), 7.63-7.71 (m, 3H), 7.24-7.62 (m, 17H), 6.5 (d,J=10.5 Hz, 1H), 4.67 (m, 1H), 4.6 (m, 1H, isomer), 4.5 (d, J=10.5 Hz,1H), 4.42 (m, 1H), 4.3 (m, 1H), 4.23 (m, 1H), 4.0 (m, 1H), 3.84 (m, 1H),2.1-2.24 (m, 3H), 1.94 (m, 1H), 1.17 (s, 9H). ¹³C NMR (CDCl₃, 150 MHz)δ:172.5, 171.3, 159.1, 158.8, 156.9, 143.8, 142.1, 141.7, 141.3, 140.8,140.4, 139.8, 130.1, 129.3, 128.9, 128.7, 127.9, 127.8, 127.7, 127.4,127.1, 127.0, 126.6, 126.5, 125.4, 125.3, 120.0, 119.9, 115.8, 113.9,67.5, 59.5, 59.3, 49.3, 47.1, 35.9, 29.7, 26.4, 25.7, 25.3.

Synthesis of tert-butylglycyl-N-phenyl-prolyl-4-amidobiphenyl, 5

Compound 4, (0.5 g, 0.74 mmol) was treated with 4.0 mL of 20% piperidinein DMF for 30 min. The reaction mixture was concentrated under vacuum.The residue was purified by to RP-HPLC (2.5×25 cm Phenomonex Luna C18column eluting with a linear gradient of MeCN in H₂O) and the purefractions then collected and lyophilized to get 0.210 g (61% yield) ofdesired material (5) as a white powder. Calcd (M+H): 456.2651; Found(M+H): 456.2708.

Synthesis of PM-242H

To a stirred solution of 5 (0.05 g, 0.1 mmol) and the active ester (6)(0.075 g, 0.1 mmol) in 3 mL of dry NMP, 40 μL of N-methylmorpholine and4-DMAP (0.002 g, 0.02 mmol) was added. The reaction was then monitoredby HPLC. After completion, the desire product then purified from thecrude by RP-HPLC (2.5×25 cm Phenomonex Luna C18 column eluting with alinear gradient of MeCN in H₂O). Combined pure fractions thenlyophilized to get of pure PM-242H as a white powder (73 mg, 71%). Calcd(M+H): 944.4063; Found (M+H): 944.4217. ¹H NMR (CDCl₃, 600 MHz) δ:7.5-7.56 (m, 4H), 7.4-7.5 (m, 7H), 7.3-7.38 (m, 3H), 7.2-7.3 (m, 5H),6.82 (d, J=10.5 Hz, 1H), 6.44 (d, J=16.0 Hz, 1H), 5.66 (m, 2H), 5.57 (m,2H), 4.8 (d, J=10.5 Hz, 1H), 3.9 (m, 1H), 3.75 (m, 1H), 1.93-2.12 (m,3H), 1.8 (m, 1H), 1.14 (s, 18H), 1.07 (s, 9H). ¹³C NMR (CDCl₃, 150 MHz)δ:176.6, 172.0, 170.3, 165.4, 140.1, 137.6, 130.0, 129.1, 128.9, 128.8,128.5, 127.8, 127.7, 127.1, 126.9, 126.5, 122.5, 82.5, 82.4, 59.0, 57.4,49.1, 38.7, 36.3, 29.8, 26.7, 26.6.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 4,764,377-   U.S. Pat. No. 5,324,756-   U.S. Pat. No. 6,426,331-   WO2001/083517-   WO2009145856A1-   WO 2001083517A1-   Blease, K. Therapeutics targeting IL-13 for the treatment of    pulmonary inflammation and airway remodeling. Curr. Opin. Investig.    Drugs 2008, 9, 1180-1184.-   Chiba, Y.; Todoroki, M.; Nishida, Y.; Tanabe, M.; Misawa, M. A novel    STAT6 inhibitor AS1517499 ameliorates antigen-induced bronchial    hypercontractility in mice. Am. J. Respir. Cell Mol. Biol. 2009, 41,    516-524.-   Corry, D. B. et al. Interleukin 4, but not interleukin 5 or    eosinophils, is required in a murine model of acute airway    hyperreactivity. J. Exp. Med. 183, 109-117 (1996).-   Darcan-Nicolaisen, Y.; Meinicke, H.; Fels, G.; Hegend, O.;    Haberland, A.; Kuhl, A.; Loddenkemper, C.; Witzenrath, M.; Kube, S.;    Henke, W.; Hamelmann, E. Small interfering RNA against transcription    factor STAT6 inhibits allergic airway inflammation and    hyperreactivity in mice. J. Immunol. 2009, 182, 7501-7508.-   Kasaian, M. T.; Miller, D. K. IL-13 as a therapeutic target for    respiratory disease. Biochem. Pharmacol. 2008, 76, 147-155.-   Kheradmand, F. et al. A protease-activated pathway underlying Th    cell type 2 activation and allergic lung disease. J. Immunol. 169,    5904-5911 (2002).-   Kuperman, D. A.; Schleimer, R. P. Interleukin-4, interleukin-13,    signal transducer and activator of transcription factor 6, and    allergic asthma. Curr. Mol. Med. 2008, 8, 384-392.-   Lee, S. H. et al. Differential requirement for CD 18 in T-helper    effector homing. Nat. Med. 9, 1281-1286 (2003).-   Mandal, P. K.; Liao, W. S.; McMurray, J. S. Synthesis of    phosphatase-stable, cell-permeable peptidomimetic prodrugs that    target the SH2 domain of Stat3. Org. Lett. 2009, 11, 3394-3397.-   Mandal, P. K.; Gao, F.; Lu, Z.; Ren, Z.; Ramesh, R.; Birtwistle, J.    S.; Kaluarachchi, K. K.; Chen, X.; Bast, R. C.; Liao, W. S.;    McMurray, J. S. Potent and Selective Phosphopeptide Mimetic Prodrugs    Targeted to the Src Homology 2 (SH2) Domain of Signal Transducer and    Activator of Transcription 3. J. Med. Chem. 2011, 54, 3549-5463.-   McCusker, C. T.; Wang, Y.; Shan, J.; Kinyanjui, M. W.; Villeneuve,    A.; Michael, H.; Fixman, E. D. Inhibition of Experimental Allergic    Airways Disease by Local Application of a Cell-Penetrating    Dominant-Negative STAT-6 Peptide. J. Immunol. 2007, 179, 2556-2564.-   Mullings, R. E.; Wilson, S. J.; Puddicombe, S. M.; Lordan, J. L.;    Bucchieri, F.; Djukanovic, R.; Howarth, P. H.; Harper, S.;    Holgate, S. T.; Davies, D. E. Signal transducer and activator of    transcription 6 (STAT-6) expression and function in asthmatic    bronchial epithelium. J. Allergy Clin. Immunol. 2001, 108, 832-838.-   Nagashima, S.; Yokota, M.; Nakai, E.; Kuromitsu, S.; Ohga, K.;    Takeuchi, M.; Tsukamoto, S.; Ohta, M. Synthesis and evaluation of    2-{[2-(4-hydroxyphenyl)-ethyl]amino}pyrimidine-5-carboxamide    derivatives as novel STAT6 inhibitors. Bioorg. Med. Chem. 2007, 15,    1044-1055.-   Nagashima, S.; Nagata, H.; Iwata, M.; Yokota, M.; Moritomo, H.;    Orita, M.; Kuromitsu, S.; Koakutsu, A.; Ohga, K.; Takeuchi, M.;    Ohta, M.; Tsukamoto, S. Identification of    4-benzylamino-2-[(4-morpholin-4-ylphenyl)amino]pyrimidine-5-carboxamide    derivatives as potent and orally bioavailable STAT6 inhibitors.    Bioorg. Med. Chem. 2008, 16, 6509-6521.-   Nagashima, S.; Hondo, T.; Nagata, H.; Ogiyama, T.; Maeda, J.;    Hoshii, H.; Kontani, T.; Kuromitsu, S.; Ohga, K.; Orita, M.; Ohno,    K.; Moritomo, A.; Shiozuka, K.; Furutani, M.; Takeuchi, M.; Ohta,    M.; Tsukamoto, S. Novel 7H-pyrrolo[2,3-d]pyrimidine derivatives as    potent and orally active STAT6 inhibitors. Bioorg. Med. Chem. 2009,    17, 6926-6936.-   Nguyen, L. P. et al. Beta2-adrenoceptor signaling is required for    the development of an asthma phenotype in a murine model. Proc Natl    Acad Sci USA 106, 2435-2440 (2009).-   Oh, C. K.; Geba, G. P.; Molfino, N. Investigational therapeutics    targeting the IL-4/IL-13/STAT-6 pathway for the treatment of asthma.    Eur. Respir. Rev. 2010, 19, 46-54.-   Ohga, K.; Kuromitsu, S.; Takezawa, R.; Numazaki, M.; Ishikawa, J.;    Nagashima, S.; Shimizu, Y. YM-341619 suppresses the differentiation    of spleen T cells into Th2 cells in vitro, eosinophilia, and airway    hyperresponsiveness in rat allergic models. Eur. J. Pharmacol. 2008,    590, 409-416.-   Polikepahad, S. et al. A reversible, non-invasive method for airway    resistance measurements and bronchoalveolar lavage fluid sampling in    mice. J. Vis. Exp. 38 (2010).-   Popescu, F. D. New asthma drugs acting on gene expression. J. Cell.    Mol. Med. 2003, 7, 475-486.-   Porter, P. et al. Link between allergic asthma and airway mucosal    infection suggested by proteinase-secreting household fungi. Mucosal    Immunol. 2, 504-517 (2009).-   Shaw, J. L. et al. IL-33-Responsive Innate Lymphoid Cells Are an    Important Source of IL-13 in Chronic Rhinosinusitis with Nasal    Polyps. Am. J. Respir. & Crit. Care Med. 188, 432-439 (2013).-   Stolzenberger, S.; Haake, M.; Duschl, A. Specific inhibition of    interleukin-4-dependent Stat6 activation by an intracellularly    delivered peptide. Eur. J. Biochem. 2001, 268, 4809-4814.-   Walker, J. K. et al. Beta-arrestin-2 regulates the development of    allergic asthma. J Clin Invest 112, 566-574 (2003).-   Walsh, G. M. An update on emerging drugs for asthma. Expert Opin.    Emerg. Drugs 2012, 17, 37-42.-   Wang, L. H.; Yang, X. Y.; Kirken, R. A.; Resau, J. H.; Farrar, W. L.    Targeted disruption of stat6 DNA binding activity by an    oligonucleotide decoy blocks IL-4-driven T(H)2 cell response. Blood    2000, 95, 1249-1257.-   Wang, Y.; Li, Y.; Shan, J.; Fixman, E.; McCusker, C. Effective    treatment of experimental ragweed-induced asthma with STAT-6-IP, a    topically delivered cell-penetrating peptide. Clin. Exp. Allergy    2011, 41, 1622-1630.-   Wu, P.; Brasseur, M.; Schindler, U. A high-throughput STAT binding    assay using fluorescence polarization. Anal. Biochem. 1997, 249,    29-36.-   Zhou, L.; Kawate, T.; Liu, X.; Kim, Y. B.; Zhao, Y.; Feng, G.;    Banerji, J.; Nash, H.; Whitehurst, C.; Jindal, S.; Siddiqui, A.;    Seed, B.; Wolfe, J. L. STAT6 phosphorylation inhibitors block    eotaxin-3 secretion in bronchial epithelial cells. Bioorg. Med.    Chem. 2012, 20, 750-758.-   Novabiochem, Guide to the Selection of Building Blocks for Peptide    Synthesis (2008)-   Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl    & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002)-   March's Advanced Organic Chemistry: Reactions, Mechanisms, and    Structure (2007)-   Remington's Pharmaceutical Sciences, 18^(th) Ed. Mack Printing    Company, pp 1289-1329, 1990.-   Remington: The Science and Practice of Pharmacy, 21^(st) Ed.    Lippincott Williams and Wilkins, 2005.

The invention claimed is:
 1. A compound of the formula:

wherein: R₂₁ is phosphate, —OP(O)(OR₁₀)(OR_(10′)),-alkyl_((C1-6))-P(O)(OR₁₀)(OR_(10′)), or a substituted version of any ofthese groups; wherein R₁₀ and R_(10′) are each independently hydrogen,alkyl_((C1-6)), aryl_((C6-8)), aralkyl_((C7-12)),alkyl_((C1-6))-O—C(O)-alkyl_((C1-6)),alkyl_((C1-6))-O—C(O)-aryl_((C6-8)), or

 wherein m=1-8; wherein X is —CH₂—, —O—, —S—, or —NH—; provided that R₁₀and R_(10′) are not both hydrogen; R₂₂ is hydrogen or alkyl_((C1-6));R₂₃ is hydrogen, alkyl_((C1-12)), or aryl_((C6-12)); R₂₄ isaryl_((C≤12)); wherein if an alkyl, aryl, or aralkyl group issubstituted then one or more hydrogen atom on the alkyl, aryl or aralkylgroup has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂,—NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —OC(O)CH₃, or —S(O)₂NH₂; or apharmaceutically acceptable salt thereof.
 2. The compound of claim 1,wherein the R₂₁ is -alkyl_((C1-6))-P(O)(OR₁₀)(OR_(10′)) or substituted-alkyl_((C1-6))-P(O)(OR₁₀)(OR)(OR_(10′)).
 3. The compound of claim 2,wherein R₂₁ is —CF₂—P(O)(OCH₂OC(O)C(CH₃)₃)₂.
 4. The compound of claim 1,wherein R₂₂ is hydrogen.
 5. The compound of claim 1, wherein R₂₃ isaryl_((C6-12)).
 6. The compound of claim 5, wherein R₂₃ is phenyl. 7.The compound of claim 1, wherein R₂₄ is aryl_((C8-C12)).
 8. The compoundof claim 7, wherein R₂₄ is biphenyl.
 9. The compound of claim 1, whereinthe compound has the formula:

or a pharmaceutically acceptable salt thereof.
 10. A pharmaceuticalcomposition comprising an effective amount of a compound of formula (I)of claim 1, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable excipient.
 11. The pharmaceuticalcomposition of claim 10, wherein the pharmaceutical composition isformulated for oral, intravenous, intranasal, or inhalationaladministration.
 12. The pharmaceutical composition of claim 11, whereinthe pharmaceutical composition is comprised in a nebulizer, an inhaler,or a nasal spray.
 13. The pharmaceutical composition of claim 12,wherein the pharmaceutical composition further comprises abronchodialator.
 14. The pharmaceutical composition of claim 13, whereinthe bronchodialator is a long-acting β2 agonist.
 15. A method oftreating an allergic or inflammatory lung disease in a subjectcomprising administering to the subject a therapeutically effectiveamount of a compound of claim 1 to the subject.
 16. The method of claim15, wherein the lung disease is asthma or airway hyperresponsiveness.17. The method of claim 15, wherein the lung disease is an allergicdisease, allergic rhinitis, emphysema, chronic obstructive pulmonarydisease (COPD), reactive airway disease, or chronic rhinosinusitis. 18.The method of claim 15, further comprising administering to the subjecta second therapeutic compound to the subject.
 19. The method of claim18, wherein the second therapeutic compound is a bronchodialator, ananti-inflammatory steroid, an antihistamine, or an anti-fungalantibiotic.
 20. The method of claim 19, wherein the second therapeuticcompound is a bronchodialator, and wherein the bronchodialator is ashort-acting β-2 agonist, a long-acting β2 agonist, or ananticholinergic.
 21. A method of inhibiting STAT6 in a subjectcomprising administering to the subject a compound of claim 1 to thesubject in an amount an effective to inhibit STAT6, wherein the subjecthas lung inflammation.
 22. The compound of claim 1, wherein R₂₃ ishydrogen or alkyl_((C1-12)).